Method for the simultaneous determination of blood group and platelet antigen genotypes

ABSTRACT

RBC and platelet (Plt) alloimmunization requires antigen-matched blood to avoid adverse transfusion reactions. Some blood collection facilities use unregulated Abs to reduce the cost of mass screening, and later confirm the phenotype with government approved reagents. Alternatively, RBC and Plt antigens can be screened by virtue of their associated single nucleotide polymorphisms (SNPs). We developed a multiplex PCR-oligonucleotide extension assay using the GenomeLab SNPStream platform to genotype blood for a plurality of blood group antigen-associated SNPs, including but not limited to: RhD (2), RhC/c, RhE/e, S/s, K/k, Kp a/b , Fya/b, FY0, Jk a/b , Di a/b , and HPA-1a/b.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation application of currentlypending U.S. Utility application Ser. No. 12/551,881, filed on Sep. 1,2009, which is a Continuation of U.S. Utility application Ser. No.10/588,631, filed Nov. 27, 2007, which is a 371 National Stage ofInternational Application No. PCT/CA2005/000250 filed on Feb. 7, 2005,which designated the U.S., and which claims benefit under 35 U.S.C.§119(e) of the U.S. provisional application Ser. No. 60/541,932, filedFeb. 6, 2004. The content of these applications is incorporated herewithin their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 26, 2014, isnamed Sequence_Listing.txt and is 7,202 bytes in size.

TECHNICAL FIELD

This invention relates to an ultra high throughput (UHT) multiplex PCRgenotyping method. More specifically, the present invention relates toan automated method of determining a plurality of blood group andplatelet antigen, preferably human platelet antigen (HPA), genotypessimultaneously from a single sample through the detection of singlenucleotide polymorphisms (SNPs) for various blood group and plateletantigens.

BACKGROUND OF THE INVENTION

At present, there are 29 blood group systems and 6 HPA systemsrecognized by the International Society of Blood Transfusion (ISBT),wherein, with a few exceptions, a blood group ‘system’ may be defined bya single gene at a given locus of the human genome (Daniels, G. L. etal. Vox Sang 2003; 84:244; Metcalfe P. et al., Vox Sang. 2003; 85:240).Most people know their ABO and Rh blood group. However, the ABO and Rhblood group systems expressed on red cells simply represent antigensfrom only two of the 29 blood group systems, and more systems are beingdiscovered each year. Some examples of blood group systems are the ABO,Rh (D, C, c, E, e), P, Lutheran, Kell (K, k), Lewis, Duffy (Fy^(a),Fy^(b)), or Kidd (Jk^(a), Jk^(b)). Moreover, there are over 250 bloodgroup and 12 human platelet antigens assigned to one of the blood groupor HPA systems, respectively. A system is defined by a gene or group ofgenes at a specific locus of the human genome. The alleles or genotypeof a person for each blood group or HPA system represent the uniquenucleotide gene sequences that express specific blood group or plateletantigens (for a review see Denomme, G. et al., Approaches to Blood GroupMolecular Genotyping and Its Applications: in Stowell, C. and Dzik W.,editors; Emerging Technologies in Transfusion Medicine, AABB 2003, Ch4).

A blood group or HPA system maps to a specific region of the humangenome, termed a locus. Nearly all blood group or HPAs can be identifiedby the presence of its unique nucleotide sequence, termed an ‘allele’,at the locus of interest. Every person has two alleles for any givenautosomal gene. Some individuals are homozygotes for a specific allele,i.e. they have two identical alleles, while others are heterozygotes fora specific allele, i.e. they have two different alleles. By definition,alleles that represent different blood group or HPAs differ by at leastone nucleotide; sometimes they differ by several nucleotides. Forexample, a deoxythymidine (T) or a deoxycytidine (C) nucleotide can befound at cDNA position 196 of the glycoprotein IIa (GP3A) gene thatexpresses the HPA-1 (Newman P. J. et al., J Clin Invest 1989; 83:;1778). The allele containing the deoxthymidine nucleotide expresses theHPA-1a antigen and the allele containing the deoxycytidine nucleotideexpresses the HPA-1b antigen. We refer to the T/C nucleotide differencebetween the two alleles as a single nucleotide polymorphism (SNP).

Blood group alleles for a given blood group system represent geneticvariations of the same gene. For example, the ABO blood group system has3 common alleles, that confer 6 genotypes within this blood groupsystem. Moreover, many alleles within a blood group system expressdifferent blood group ‘antigens’, that is to say, dependent on theallelic genotype the corresponding antigenic phenotype is accordinglyexpressed. Alleles differ in their nucleotide sequence, and thedifference between one allele and another, usually within a single bloodgroup system, may be one single nucleotide variation. Therefore, twoalleles can differ by one nucleotide, i.e. a SNP and represent aco-dominant bi-allelic system. Alternatively, alleles can differ by afew to several dispersed nucleotides, or by a stretch of nucleotides,any one of which can be used to identify the alleles. Regardless ofwhether the variations in the nucleotide are due to single or multiplenucleotide differences, the phenotype associated with a specificgenotype (the specific nucleotide sequence) will result in theexpression of a specific blood group or platelet antigen on the red cellor platelet surface, respectively.

Normally, all blood donations are blood grouped for ABO and RhD.However, sometimes a previously transfused recipient will require moreblood that is antigen-matched with one of their own antigens becausethey have made antibodies to a different blood group or plateletantigen. The gold standard in the industry is to ‘phenotype’ blood forthe presence of specific blood group and platelet antigens usinggovernment regulated antisera (antibodies) performed by single-testmethods or by an automated platform, which is a cost ineffective methodfor a blood collection facility that routinely performs tests on a highvolume basis.

Blood group phenotypes are presently determined using commerciallyavailable government-regulated serological reagents and human red cells.These known tests rely on the principle of antibody binding and red cellagglutination to identify clinically important blood group phenotypes.The presently known tests were originally devised some 60 years ago andtoday require the use of government regulated (for example, HealthCanada) approved serological reagents. Some of the tests being employedtoday have been automated (for example, ABO and Rh typing) while somehave been semi-automated (for example, RhC/c and RhE/e). However, manyof the presently used tests are performed manually by highly-trainedlaboratory technologists and are done on a test-by-test basis. In otherwords, a technologist must perform four separate tests to determine, forexample, the Fy^(a), Fy^(b), Jk^(a) and Jk^(b) phenotype of a singleblood donation. Essentially, the current tests which employgovernment-approved reagents in a manual, single-test driven method area very cost ineffective method for a blood collection facility that isoften required to perform such tests on a high volume basis.

In an effort to reduce costs, a blood collection facility will often usenon-regulated antisera to ‘screen’ blood donations for important bloodgroup phenotypes and then confirm the phenotype with the regulatedantisera. However, since much of the blood is sent to hospitals within24-48 hours after collection, manual blood group phenotyping cannot meetthe short turn-around time required to provide the end user with theinformation required before blood must be shipped. Therefore, hospitalblood banks must perform their own tests on the blood that they have intheir inventories. It would be advantageous to provide a cost effectiveblood screening method that would provide quick and reliable resultsrelating to the clinically important blood group phenotypes.

The prior art uses two basic techniques to detect SNPs; polymerase chainreaction-restriction fragment length polymorphism (PCR-RFLP) (ChaudhuriA., et al. 1995; 85:615), and sequence specific primer (SSP)-PCR(McFarland J. G. et al., Blood 1991; 78:2276). For PCR-RFLP analysis,restriction enzymes are used to digest PCR amplified genomic DNAfragments. In brief, DNA is extracted from nucleated blood cellsmanually for each blood sample to be analyzed. The PCR is set upmanually; a separate PCR is performed on each sample for each SNP ofinterest. The PCR amplified fragments are digested with a specificrestriction enzyme and the digested products are separated on a gel. Thepattern of digested DNA fragments viewed from the gel predicts thepresence or absence of either nucleotide of a SNP of interest. InSSP-PCR, two PCRs are set up in separate tubes for each SNP of interest.One tube contains a universal primer and a primer with a sequence thatis specific to detect one nucleotide of a SNP. The other tube containsthe same universal primer and a primer specific for the other nucleotideof a SNP. Prior art has used two pair or three pair PCR to analyze anucleotide for a given SNP, with at least one pair acting as an internalcontrol to ensure DNA is available for PCR amplification. The prior artdoes not provide the use of multiple DNA sequences as primer pairs thatwork simultaneously on a single sample. Moreover, the prior art does notemploy novel DNA sequences to detect blood group SNPs in an automatedhigh-throughput fashion.

St-Louis M., et al. (Transfusion 2003; 43:11126-32) have usedallele-specific PCR-ELISA to detect blood group SNPs, wherein some ofthe PCR primers were publicly known and all primers were labeled withdigoxigenin; SNPs were detected by oligonucleotide hybridization usingsolid-phase microplate wells coated with individual blood group-specificcomplementary oligonucleotides. An abstract by Buffleir E. et al.(Transfusion 2003; 43:92 A) outlines a combined HPA-1 and HPA-5genotyping method that uses biotin labeled PCR-amplified targets andallele specific oligonucleotide probes arrayed on the bottom of 96 wellmicroplates. Specific hybridization is detected with the use of anenzyme conjugate which produces a specific colourimetric signal. Anarray of several oligonucleotides reportedly can be used to detect HPASNPs. The publications, cited above, do not use multiplex PCR primers,nor do they use extension probes, and rely on a less sensitive and moreerror-prone allele-specific hybridization to detect the SNPs. There area few other publications that refer to the multiplex PCR amplificationof the RHD gene alone, or together with sex determination, or withinternal control primers designed to confirm the presence of DNA invarious blood group PCR applications. U.S. Pat. No. 5,723,293 describesa diagnostic method and kit for determining Rh blood group genotypes,wherein there is provided a method for directly determining D andassociated CcEe genotypes using restriction fragment lengthpolymorphisms (RFLPs) for diagnosis. U.S. Pat. No. 5,804,379 describes adiagnostic method and kit for determining Kell blood group genotype,wherein there is provided a method for determining the K1/K2 genotypeusing RFLPs for diagnosis. U.S. Pat. No. 5,780,229 providespolynucleotides for determining the Pen polymorphism of human plateletmembrane glycoprotein IIa, and generally describes diagnostic andtherapeutic uses relating to the “Pen” human platelet polymorphism(HPA-4) and differs from the teachings of the present invention. UnitedStates patent application 20020098528 describes methods and apparatusfor blood typing with optical bio-disc, and essentially describes amethod for determining the ABO blood cell type of an individual withoptical bio-discs and a disc-reading apparatus.

In the SSP-PCR application by St. Louis et al. (Transfusion 2003;43:1126), two PCR primer pairs are set up, each in a separate well, todetect the nucleotides of a SNP of interest. For example, one primerpair containing a universal primer and a sequence specific primer is setup in a tube to detect a nucleotide of a SNP. Another primer paircontaining the same universal and another sequence specific primer isset up in another tube to detect the alternate nucleotide for the sameSNP. In addition, each tube includes a primer pair that detects auniversal sequence contained in all human DNA. Contained in the PCR tubeis digoxigenin-dUTP that is incorporated into the amplified DNA fragmentif the sequence specific primer detects the appropriate nucleotide of anSNP. For the detection phase, one of each primer pair contains thechemical tag biotin, which is used to capture the DNA amplified fragmentin sets of microtitre wells containing streptavidin. An opticalcolorimetric assay is used to detect the presence of digoxigenin-dUTP ineach of the wells; anti-digoxigenin peroxidase conjugated antibodydetects the presence of digoxigenin dUTP and the peroxidase can converta substrate added to the well into a colored end product. Therefore, thepresence of a nucleotide of a SNP is detected by the presence of a colorin the microtitre well. Such assays are routinely designed in a 96-wellmicrotitre plate format to facilitate semi-automation. The colorimetricresults are evaluated by the operator to determine the presence orabsence of the nucleotides for a SNP. The deficiencies of these testsystems are the use of a single PCR reaction for each nucleotide of agiven nucleotide of each SNP, and the pooling of samples prior to thedetection phase and manual post-analyte data analysis.

No prior art has used a multiple, or 12, primer pair multiplexed PCRthat successfully works in a single tube, nor has prior art employednovel DNA sequences as probes to detect both nucleotides of a pluralityof blood group and HPA genotypes simultaneously, such as the detectionof all 12 blood group and HPA SNPs in these mixtures using an automatedhigh-throughput platform.

Accordingly, there is a need for a high-throughput automated multipleblood-group associated SNP analysis of genomic DNA that is capable ofrapidly and accurately determining the genotypes and associatedphenotypes of a plurality of blood group systems in a single testsample.

SUMMARY OF THE INVENTION

The present invention provides a method of detecting the presence orabsence of nucleotides relating to various SNPs for the determination ofa specific genotype and accordingly the inferred phenotype. Morespecifically, the present invention allows for the detection of thepresence or absence of two nucleotides of a plurality of different SNPs,and more preferably of the 12 SNPs in a preferred embodiment of thepresent invention.

The present invention accordingly provides an automated, or robotic,high-throughput ‘screening’ tool for blood group and platelet antigensby evaluating the alleles of the genes that express these antigens onred cells and platelets, respectively. This is done by identifying theunique nucleotides associated with the specific alleles that occupy thegene locus using a testing platform, which requires novel and specificcompounds that we designed. Our robotic high-throughput platformprovides important blood group and HPA genotype information within 24hours from the start of the test. We identified the alleles of bloodgroup antigens for; RhD, RhC, Rhc, RhE, Rhe, S, s, Duffy (Fy)^(a),Fy^(b), K, k, Kp^(a), Kp^(b), Diego (Di)^(a), Di^(b), Kidd (Jk)^(a),Jk^(b), and the platelet antigens, Human Platelet Antigen (HPA)-1a andHPA-1b, representing, but not limited to 19 of the most clinicallyimportant antigens in red cell and platelet transfusion. Additionalgenotyping tests for other clinically important blood group and plateletantigens may be developed, and are encompassed in the teachings of thepresent invention. When performed on all blood donations for allclinically important blood group and platelet antigens, our inventionwill provide a comprehensive database to select and confirm the antigenswhen required using government regulated antisera. The use of thisplatform as a screening tool will lessen the number of costly governmentregulated tests to be done by the collection facility and end user (thehospital blood bank), and meet the demand of antigen-matched blood forspecific transfusion recipients.

The invention discloses a method for DNA-based blood group genotypingfor clinically important blood group and platelet antigens. Thetechnology uses an ultra high-throughput multiplex PCR design to detectspecific SNPs that represent clinically important blood group antigens:RhD, RhC, Rhc, RhE, Rhe, S, s, Duffy (Fy)^(a), Fy^(b), K, k, Kp^(a),Kp^(b), Diego (Di)^(a), Di^(b), Kidd (Jk)^(a), Jk^(b), and the plateletantigens, Human Platelet Antigen (HPA)-1a and HPA-1b. It should be notedhowever that the present invention is not limited to the detection ofSNPs for only the SNPs listed, but additionally comprises the detectionof SNPs for all blood group and platelet antigens. The inventiondiscloses novel DNA sequences of PCR primers that are specificallydesigned to avoid inter-primer pair cross-reactions and post-PCR probesthat make multiple analyses possible. The invention represents a novelapproach to screening multiple blood group and HPA genotypes at once andaddresses a clear need in the art for novel, rapid, cost-effective andreliable genotyping. This additionally replaces the use of expensive anddifficult-to-obtain serological reagents, which can be reserved for useto confirm only the donors identified by the screening process.

More specifically, the present invention analyzes the HPA-1 GP3Amutation incorporated into our SNP assay, and the other blood groupantigen SNPs in a method according to the present invention.

The invention addresses the need for an automated, accurate, rapid andcost-effective approach to the identification of multiple blood groupantigens. The multiplex SNP assay design and automated genotypingplatform allows one trained research technician to identify a pluralityof blood group alleles, and more specifically, 19 blood group alleles,overnight on 372 to 2232 individual blood samples. In one application ofthe present invention, the multiplex PCR and SNP detection platformanalyzed the nucleotides of 12 SNPs overnight on 372 individual bloodsamples. The cost using current standard blood group serology for 372samples is estimated at CDN$99,500, which reflects a reagent cost ofCDN$54,000 (excluding new capital equipment investments) and an operatorcost of CDN$45,500 to analyse each of the antigens by Gel Cardtechnology (n=5), immediate spin tube test (n=2), indirect antiglobulintube test (n=8), and platelet GTI® test (n=1). Approximate 10 to 15 foldcost savings are obtained in the simultaneous DNA-based determination ofthese blood group alleles. It should be noted that the present inventionis not limited to the detection of only 12 SNPs, and may be optimallyused for the detection a plurality of SNPs for potentially all bloodgroup and platelet alleles. Accordingly, the products, methods, platformand teachings of the present invention can detect all blood group andHPA SNP variations on a great number of samples, such as 744 samplesovernight, as further described below.

The present invention overcomes the deficiencies of the prior artbecause the entire test, i.e. all steps of the method of the presentinvention, from PCR to computation analyses can be automated andmultiplexed so that the nucleotides of a plurality of SNPs, and morepreferably, the 12 SNPs of the present invention, can be identifiedsimultaneously. This automated multiplex high throughput analysis canmeet the demand of testing hundreds of blood samples, and theturn-around time of less than 24 hours, to provide valuable informationto a blood collection facility before blood is shipped to the end user.This platform has the advantage over existing technology in that itreduces operator handling error. In addition, there are significant costreductions compared with the current government-regulated serologicalanalysis. It should be noted that present prior art technologiesrelating to PCR-RFLP and SSP-PCR for blood group and platelet antigensare not routinely used since they are no more cost efficient thanserology. The present invention overcomes the deficiencies of the priorart and fulfils an important need in the present art for the automated,accurate, rapid and cost-effective identification of multiple bloodgroup and HPA SNPs.

The invention provides the opportunity to screen all blood donors toobtain a daily or ‘live’ repository of the genotypes or combinations ofgenotypes currently available for specific transfusion needs.Accordingly, the present invention fulfills a need relating to thecollection and antigen screening of blood and blood products.

For convenience, some terms employed in the present specification arenoted below. Unless defined otherwise, all technical and scientificterms used herein have the meanings commonly understood by one ofordinary skill in the present art.

The present invention provides a method or screening assay for thedetermination of blood genotypes of the various blood group and HPAsystems through the ultra high throughput multiplex PCR analysis of SNPsin an automated platform (Petrick J. Vox Sang 2001; 80:1). A platform,as referred to herein, refers to a system of machine(s) and protocol(s)capable of analyzing multiplex PCR amplified SNPs, wherein said platformis not limited to, but may comprise the GenomeLab SNPStream (BeckmanCoulter Inc., Fullerton, Calif.), the SNPSTREAM™ UHT (OrchidBioSciences, Princeton, N.J.), the SNPSTREAM™ 25K (Orchid BioSciences,Princeton, N.J.), the MALDI-TOF/Mass-Spectrophotometer Spectro CHIP(Sequenom, San Diego, Calif.), and the Gene Chip Microarray (Affymetrix,Inc., Santa Clara, Calif.), Nano Chip (Nanogen, San Diego, Calif.) andthe Random Ordered Bead Arrays (Illumina, Inc., San Diego, Calif.) orany other system, machine or protocol capable of analyzing multiplex PCRamplified SNPs. Accordingly, the present invention provides a platform,or system and protocols, for the evaluation and detection of SNPs, forthe purpose of typing (determining the genotype and correspondingphenotype) blood group and platelet, preferably, human platelet antigen(HPA) SNP analysis. A preferred platform that can be used in accordancewith the present invention is the Orchid SNP-IT system for HLA typing(Orchid Bioscience, Princeton, N.J.), wherein a preferred embodiment ofthe present invention comprises the use of the primer pairs of Table 1for the specific oligonucleotide primer extension of blood group andplatelet, preferably, human platelet antigen (HPA) SNPs, and the probesof Table 2 for the specific hybridization thereof, and the simultaneousanalysis of the absence or presence of a plurality of blood group andplatelet, preferably, human platelet antigen (HPA) SNPs using a platformas described herein, or using any SNP analysis system capable ofdetecting multiplex PCR amplified SNPs.

For the purposes of the present disclosure, SNPs, may refer to any bloodgroup and HPA SNPs, and more preferably refers to any of the SNPsspecified in Table 1, or any other known blood group or HPA SNPs orsingle nucleotide changes including, but not limited to, nucleotidesubstitutions, deletions, insertions or inversions, that can be definedas a blood group or HPA SNP due to nucleotide differences at thespecified position in a gene sequence.

Ultra high throughput (UHT) refers to the implementation of the platformin a rapid and optimized form, that is to say, through the analysis ofmultiple SNPs. That is to say, UHT analysis refers to the rapid andsimultaneous evaluation of a plurality of samples for a plurality ofmarkers, in this case SNPs. For example, the analysis of 12 SNPs(equivalent to 12 C and 12 T nucleotides) for 372 samples, would resultin the generation of 8928 (i.e. 2×12×372) determinations that areanalysed, an evaluation that far exceeds the number of evaluation pointspossible with manual or automated serological methods.

Phenotype in the context of red cell blood group and Human PlateletAntigen (HPA) refers to the expressed moiety of an allele for a givengene, and is also referred to in this document as ‘antigen’. Genotyperefers to the two alleles of an autosomal gene that occupy a given locusor alternatively to either one or two alleles of an X-linked gene thatoccupies a given locus.

Antigen refers to a red cell or platelet membrane carbohydrate, proteinor glycoprotein that is expressed as a polymorphic structure among thehuman population, that is to say a moiety that is immunogenic in anotheranimal, or human, due differences in its amino acid or carbohydratecomposition. Blood group or red cell, or HPA or platelet antigen refersto a moiety expressed on red cells or platelets that has been assigned ablood group or Human Platelet Antigen (HPA) designation, or provisionalor workshop designation. The present invention comprises a method andfor the determination of the antigen genotype and correspondingphenotype of any blood group or red cell, or HPA or platelet antigenusing multiplex PCR SNP analysis. The following two tables (Table A andTable B) list most of the known human blood group and platelet antigens.Many of the antigens can be identified by their unique nucleotidesequence.

TABLE A Human Red Cell Blood Group Systems Component Associated ISBTName (ISBT Chromosome Gene Name Name Blood Group Number) Location ISGN(ISBT) (CD Number) Antigens ABO (001) 9q34.2 ABO (ABO) Carbohydrate A,B, A, B, A1 MNS (002) 4q28.2-q31.1 GYPA (MNS) GPA (CD235a) M, N, Vw,GYPB (MNS) GPB (CD235b) S, s, U, He + 36 more P (003) 22q11.2-qter P1(P1) Carbohydrate P1 Rh (004) 1p36.13-p34.3 RHD (RH) RhD (CD240D) D, G,Tar RHCE (RH) RhCE C, E, c, e, V, (CD240CE) Rh17 + 39 more Lutheran(005) 19q13.2 LU (LU) Lutheran glyco- Lu^(a), Lu^(b), Lu3, protein Lu4,Au^(a), Au^(b) + B-CAM (CD239) 13 more Kell (006) 7q33 KEL (KEL) Kellglycoprotein K, k, Kp^(a), Kp^(b), (CD258) Ku, Js^(a), Js^(b) + 17 moreLewis (007) 19p13.3 FUT3 (LE) Carbohydrate Le^(a), Le^(b), Le^(ab),Adsorbed form Le^(bh), ALe^(b), BLe^(b) plasma Duffy (008) 1q22-q23 DARC(FY) Fy glycoprotein Fy^(a), Fy^(b), Fy3, (CD234) Fy4, Fy5, Fy6 Kidd(009) 18q11-q12 SLC14A1 (JK) Kidd Jk^(a), Jk^(b), Jk3 glycoprotein Diego(010) 17q21-q22 SLC4A1 (DI) Band 3, AE1 Di^(a), Di^(b), Wr^(a), (CD233)Wr^(b), Wd^(a), Rb^(a) + 14 more Yt (011) 7q22 ACHE (YT) Acetyl- Yt^(a),Yt^(b) cholinesterase Xg (012) Xp22.32 XG (XG) Xg^(a) glycoproteinXg^(a) MIC2 CD99 CD99 Scianna (013) 1p34 ERMAP (SC) ERMAP Sc1, Sc2, Sc3,Rd Dombrock (014) 12p13.2-p12.1 DO (DO) Do glycoprotein; Do^(a), Do^(b),Gy^(a), ART 4 Hy, Jo^(a) Colton (015) 7p14 AQP1 (CO) Channel-formingCo^(a), Co^(b), Co3 integral protein Landsteiner- 19p13.3 LW (LW) LWglycoprotein LW^(a), LW^(ab), LW^(b) Wiener (016) (ICAM-4) (CD242)Chido/Rodgers 6p21.3 C4B, C4A C4B, C4A CH1, CH2, Rg1 + (017) (CH/RG) 6more Hh (018) 19q13.3 FUT1 (H) Carbohydrate H (CD173) Kx (019) Xp21.1 XK(XK) Xk glycoprotein Kx Gerbich (020) 2q14-q21 GYPC (GE) GPC Ge3, Ge4,Wb, GPD (CD236) Ls^(a), Dh^(a) Ge2, Ge3, An^(a) Cromer (021) 1q32 DAF(CROM) DAF (CD55) Cr^(a), Tc^(a), Tc^(b), Tc^(c), Dr^(a), Es^(a), IFC,WES^(a), WES^(b), UMC, GUTI Knops (022) 1q32 CR1 (KN) CR1 (CD35) Kn^(a),Kn^(b), McC^(a), Sl^(a), Yk^(a) Indian (023) 11p13 CD44 (IN) Hermesantigen In^(a), In^(b) (CD44) OK (024) 19pter-p13.2 CD147 (OK)Neurothelin, Ok^(a) basogin (CD147) RAPH (025) 11p15.5 MER2 (MER2) Notdefined MER2 JMH (026) 15q22.3-q23 SEMA-L (JMH) H-Sema-L JMH (CD108) I(027) 6p24 CGNT2 (IGNT) Carbohydrate I Globoside (028) 3q25 B3GALT3Carbohydrate P (βGalNAcT1) (Gb₄, globoside) GIL (029) 9p13 AQP3 (GIL)AQP3 GIL ISGN = International Society for Gene Nomenclature

TABLE B Human Platelet Antigen Systems Chromo- Gene some AssociatedSystem Name Location Component Name (CD) Antigens HPA-1 GP3A 17q21.32Integrin β3 (CD61) Pl^(A1/2) HPA-2 GP1BA 17pter- Glycoprotein IbαKo^(a/b) p12 (CD42b) HPA-3 GP2B 17q21.32 Integrin α2b (CD41) Bak^(a/b)HPA-4 GP3A 17q21.32 Integrin β3 (CD61) Pen^(a/b) HPA-5 GP1A 5q23-q31Integrin α2 (CD49b) Br^(a/b) HPA-6w GP3A 17q21.32 Integrin β3 (CD61)Ca^(a)/Tu^(a) HPA-7w GP3A 17q21.32 Integrin β3 (CD61) Mo^(a) HPA-8w GP3A17q21.32 Integrin β3 (CD61) Sr^(a) HPA-9w GP2B 17q21.32 Integrin α2b(CD41) Max^(a) HPA- GP3A 17q21.32 Integrin β3 (CD61) La^(a) 10w HPA-GP3A 17q21.32 Integrin β3 (CD61) Gro^(a) 11w HPA- GP1BB 22q11.2Glycoprotein Ibβ Ly^(a) 12w (CD42c) HPA- GP1A 5q23-q31 Integrin α2(CD49b) Sit^(a) 13w HPA- GP3A 17q21.32 Integrin β3 (CD61) Oe^(a) 14wHPA-15 AF410459 6q13 GPI-linked GP (CD109) Gov^(a/b) HPA- GP3A 17q21.32Integrin β3 (CD61) Duv^(a) 16w ? GPV ? Glycoprotein V Pl^(T) ? GPIV7q11.2 Glycoprotein IV (CD36) Vis^(a)/Nak^(a) Note: HPA numbers on theleft ending with a ‘w’ represent ISBT workshop designations and aretentative HPA systems.

A single nucleotide polymorphism (SNP) refers to any blood group or HPAallele that defines a specific red cell or platelet antigen by virtue ofits unique nucleotide sequence as defined in Garratty et al. Transfusion2000; 40:477 and as updated from time-to-time by the InternationalSociety of Blood Transfusion.

It is understood that the presently disclosed subject matter is notlimited to the particular methodology, protocols, cell lines, vectors,and reagents described as these can vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe presently disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinare intended to have their meanings as understood by one skilled in thepresent art. Although any methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently disclosed subject matter, the preferred embodiments, methods,devices and materials described.

It is also understood that the articles ‘a’ and ‘an’ are used herein torefer to one or to more than one (i.e. to at least one) of thegrammatical object of the article. Accordingly, ‘an element’ means oneelement or more than one element.

Our novel platform simultaneously performs automated multiple bloodgroup-associated SNP analyses using genomic DNA and the Thermusaquaticus polymerase chain reaction (PCR) to infer the presence ofspecific blood group genotypes. This automated high-throughput platformhas particular application in the blood donation industry since itrepresents a novel screening tool for the expression of blood groupantigens or phenotypes.

Our platform provides important genotypic information within 24 hours ofdonation. When performed on all blood donations for all important bloodgroup phenotypes, our invention will provide a comprehensive database toselect and confirm blood group phenotypes using government regulatedantisera. The use of this platform as a screening tool will lessen thenumber of regulated blood group phenotype tests done by the collectionfacility and end user, and meet the end user demand for antigen-matchedblood for transfusion recipients.

Unique to this invention is the assay design for the simultaneousidentification of a plurality of blood group or HPA alleles. The presentinvention provides novel assay for the simultaneous identification of aplurality of blood group or HPA alleles, and more preferably of 19 bloodgroup alleles using a plurality of SNPs, and more preferably, 12 SNPs.In one embodiment, the genotyping platform queries genetic variantsusing multiplexed single nucleotide primer extension coupled withtwo-laser fluorescence detection and software for automated genotypecalling. Each of the relevant gene regions are PCR amplified frompurified genomic DNA in a single reaction using the followingoligonucleotide primer designs:

Gene Primer Sequence (5′ - 3′) RHD Exon RHDe4S AGACAAACTGGGTATCGTTGC 4(SEQ ID NO: 1) RHDe4A ATCTACGTGTTCGCAGCCT (SEQ ID NO: 2) RHD Exon RHDe9SCCAAACCTTTTAACATTAAATTATGC 9 (SEQ ID NO: 3) RHDe9ATTGGTCATCAAAATATTTAGCCTC (SEQ ID NO: 4) RHCE Exon RHCEe2STGTGCAGTGGGCAATCCT (SEQ ID NO: 2 5) RHCEe2ACCACCATCCCAATACCTG (SEQ ID NO: 6) RHCE Exon RHCEe5SAACCACCCTCTCTGGCCC (SEQ ID NO: 5 7) RHCEe5A ATAGTAGGTGTTGAACATGGCAT(SEQ ID NO: 8) GYPB Exon GYPBe4S ACATGTCTTTCTTATTTGGACTTAC 4(SEQ ID NO: 9) GYPBe4A TTTGTCAAATATTAACATACCTGGTAC (SEQ ID NO: 10)KEL Exon KELe6S TCTCTCTCCTTTAAAGCTTGGA 6 (SEQ ID NO: 11) KELe6AAGAGGCAGGATGAGGTCC (SEQ ID NO: 12) KEL Exon KELe8S AGCAAGGTGCAAGAACACT 8(SEQ ID NO: 13) KELe8A AGAGCTTGCCCTGTGCCC (SEQ ID NO: 14) FY FYproSTGTCCCTGCCCAGAACCT (SEQ ID NO: Promoter 15) FYproAAGACAGAAGGGCTGGGAC (SEQ ID NO: 16) FY Exon FYe2S AGTGCAGAGTCATCCAGCA 2(SEQ ID NO: 17) FYe2A TTCGAAGATGTATGGAATTCTTC SEQ ID NO: 18) JK ExonJKe9S CATGAACATTCCTCCCATTG 9 (SEQ ID NO: 19) JKe9ATTTAGTCCTGAGTTCTGACCCC (SEQ ID NO: 20) DI Exon DIe19SATCCAGATCATCTGCCTGG 18 (SEQ ID NO: 21) DIe19ACGGCACAGTGAGGATGAG (SEQ ID NO: 22) GP3A GP3Ae3S ATTCTGGGGCACAGTTATCC(SEQ ID NO: 23) GP3Ae3A ATAGTTCTGATTGCTGGACTTCTC (SEQ ID NO: 24)

The above primer pairs comprise the corresponding forward and reverseprimers, and may be referred to herein as SEQ ID NOs 1-24.

Multiplexed single nucleotide primer extension is performed using thefollowing 5′ tagged extension primers:

RHD Exon 4 (SEQ ID NO: 25) GTGATTCTGTACGTGTCGCCGTCTGATCTTTATCCTCCGTTCCCTRHD Exon 9 (SEQ ID NO: 26) GCGGTAGGTTCCCGACATATTTTAAACAGGTTTGCTCCTAAATCTRHCE Exon 2 (SEQ ID NO: 27)GGATGGCGTTCCGTCCTATTGGACGGCTTCCTGAGCCAGTTCCCT RHCE Exon 5(SEQ ID NO: 28) CGACTGTAGGTGCGTAACTCGATGTTCTGGCCAAGTGTCAACTCTGYPB Exon 4 (SEQ ID NO: 29) AGGGTCTCTACGCTGACGATTTGAAATTTTGCTTTATAGGAGAAA KEL Exon 6 (SEQ ID NO: 30)AGCGATCTGCGAGACCGTATTGGACTTCCTTAAACTTTAACCGAA KEL Exon 8 (SEQ ID NO: 31)AGATAGAGTCGATGCCAGCTTTCCTTGTCAATCTCCATCACTTCA FY Promoter(SEQ ID NO: 32) GACCTGGGTGTCGATACCTAGGCCCTCATTAGTCCTTGGCTCTTA FY Exon 2(SEQ ID NO: 33) ACGCACGTCCACGGTGATTTGGGGGCAGCTGCTTCCAGGTTGGCA JK Exon 9(SEQ ID NO: 34) CGTGCCGCTCGTGATAGAATAAACCCCAGAGTCCAAAGTAGATGT DI Exon 19(SEQ ID NO: 35) GGCTATGATTCGCAATGCTTGTGCTGTGGGTGGTGAAGTCCACGCGP3A Exon 3 (SEQ ID NO: 36) AGAGCGAGTGACGCATACTTGGGCTCCTGTCTTACAGP3A Exon 3 (SEQ ID NO: 37) GCCCTGCCTC

The above probes may be referred to herein as SEQ ID NOs 25-37. The DNAbases are represented by their single letter equivalents (A,C,G or T)and SEQ ID NOs: 36 and 37 are joined together by a C3 (phosphoramidite)spacer between the two sequences represented by letter X as followsAGAGCGAGTGACGCATACTTGGGCTCCTGTCTTACAXGCCCTGCCT (SEQ ID NO. 36 X SEQ IDNO. 37).

In this embodiment, the 12 bolded nucleotides in the 5′ region of theextension probes are hybridized to a complementary DNA sequence that hasbeen micro-arrayed onto microplates so that specific blood group SNPsare individually identified and reported.

Proof of principle experiments have been performed using 372 consentqualified samples (please refer to Appendix A). Collection ofserological data for samples has been constant and the success ratesbased upon the expected allele frequencies have been performed.

In the preceding example, one preferred embodiment has been described.However, it should be obvious to one skilled in the art that othermethodologies and/or technologies for SNP identification could be used,providing that the novel DNA sequences disclosed above are also used.

The teachings and method of the present invention are superior to theteachings of the prior art for a number of reasons, one of which is thatthe complete method of the present invention, from DNA extraction toresult computation analyses can be automated and multiplexed so thatmany SNPs can be determined simultaneously. This automated multiplexhigh throughput analysis can meet the demand (hundreds of blooddonations can be tested) and the turn-around time (<24 hours) to collateand provide valuable information to the blood collection facility beforeblood is shipped to the end user. This platform and method has thefurther advantage over existing technology in that it reduces operatorhandling error.

In addition, there are significant cost reductions compared with thecurrent technology. The invention addresses the need for an automated,accurate, rapid and cost-effective approach to the identification ofmultiple blood group SNPs. According to an embodiment, a multiplex SNPassay of the present invention detected 12 SNPs overnight on 372individual blood samples. In accordance with the teachings of thepresent invention, the platform, products and methods of the presentinvention can detect all SNP variations for all blood group antigens,for example, as shown below on 744 samples.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the present invention will becomeapparent from the following detailed description, taken in combinationwith the appended drawings, in which:

FIG. 1 A computer screen display of a typical UHT SNP scatter plot tosort the fluorescence of a C/T SNP analysis of GP3A Exon 3 for HPA-1a/bgenotyping.

FIG. 2 Representative samples of GP3A Exon 3 HPA-1a/b) genotyping bymanual PCR-RFLP analysis using MspI restriction enzyme analysis (A) andthe tabulated comparative results with the UHT SNP analysis (B).

FIG. 3 Representative samples JK genotyped by manual PCR-RFLP analysisusing MnlI (A) and the tabulated comparative results with the UHT SNPanalysis (B).

FIG. 4 A-L Computer screen displays of typical UHT SNP scatter plots tosort the fluorescence of a C/T SNP for various blood group and HPAgenotypes.

Appendix A provides a tabulated summary of the multiplex SNP assaydetection of 12 possible SNPs on 372 individual blood samples.

It will be noted that throughout the appended drawings, like featuresare identified by like reference numerals.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

RBC and platelet (Plt) alloimmunization requires antigen-matched bloodto avoid adverse transfusion reactions. Some blood collection facilitiesuse unregulated Abs to reduce the cost of mass screening, and laterconfirm the phenotype with government approved reagents. Alternatively,RBC and Plt antigens can be screened by virtue of their associatedsingle nucleotide polymorphisms (SNPs). The present invention provides amultiplex PCR-oligonucleotide extension assay using the GenomeLabSNPStream platform, or any other SNP analysis system, to genotype bloodfor a plurality of common antigen-associated SNPs, including but notlimited to: RhD (2), RhC/c, RhE/e, S/s, K/k, Kp^(a/b), Fya/b, FY0,Jk^(a/b), Di^(a/b), and HPA-1a/b. According to one example of thepresent invention, a total of 372 samples were analysed for 12 SNPsovernight. Individual SNP pass rates varied from 98-100% for 11 of 12SNPs. Of the Rh-pos, 98.6% were correctly identified. Six of 66 Rh-neg(9%) were typed as RHD-pos; 5 of 6 were subsequently demonstrated tocontain an non-RHDψ gene by SSP-PCR. Eleven of 12 R1R1 and 1 of 1 r″rwere correctly identified. HPA-1b was identified in 4, which wasconfirmed by PCR-RFLP (n=4) and serology (n=1). PCR-RFLP on selectedsamples (n<20) for K/k, Fy^(a/b), and Jk^(a/b) were 100% concordant.Confirmation of some of the results is provided. The platform has thecapacity to genotype thousands of samples per day for all SNPvariations. The suite of SNPs can provide collection facilities withreal-time genotypic data for all donors at an annual cost (excludingRhD) estimated to equal the current cost of phenotyping 5-10% of thedonors.

Methods and Reagents Methodology Specific to the Invention.

We have designed a novel blood group and HPA SNP and detection systemthat employ the use of two sets of novel compounds (reagents) that arespecifically designed to work in a multiplex format.

In brief, genomic DNA is harvested the salting out procedure using theQiagen (Qiagen Inc. Valencia, Calif.) Blood DNA Isolation Kit. Ourinvention can use any good quality DNA harvested by any one of a varietyof methods. For the multiplex PCR, the DNA regions containing all 12SNPs of interest were PCR-amplified in a single reaction well. Tables 1and 2 outline the novel PCR primers and extension probes, respectively,used in the assay. Note that the concentration of the various reagentsmay be adjusted to optimize DNA amplification, and is dependent on butis not limited to: the concentration and quality of the genomic DNA, theconcentration of the PCR primers or the type of thermal cycler used forthe PCR.

Our current genotyping technology identifies SNPs using single base-pairprimer extension using the novel products and protocols of the presentinvention. In brief, the genomic region surrounding the SNP of interestis PCR-amplified as described above, preferably using one or more, orall of the primer pairs of Table 1. Then, the amplified DNA fragmentsare used as a template for DNA hybridization using one or more or allthe corresponding novel probes of Table 2, and single nucleotideextension (synthesis) based on the nucleotide present at each of thespecific SNP sites. The PCR primers pairs in Table 1 represent sequencescomplementary to DNA regions containing SNPs of interest; of which theexact sequences of each primer pair and mixture of primer pairs havebeen specifically optimized to amplify genomic DNA of interest as amixture of 12 primer pairs. Although noted above, Table 2 furthersummarizes 12 novel extension primers specifically used together todetect the nucleotides of blood group and platelet antigen or HPA SNPs,simultaneously. The extension primers represent a group of 12 novelnucleotide sequences, of which each are a combination of: 1) a unique 5′region necessary to direct hybridization to a micro-arrayed tag locatedin a specific spot in each microplate well, and 2) a 3′ regioncomplementary to and adjacent to a SNP of a PCR-amplified DNA regioncontaining the SNP of interest.

TABLE 1The PCR primers used in the 12-pair multiplex PCR format for multiple SNPdetection. Primer Product Size Antigen SNP Name Sequence 5′-3′ Target(bp) RhD/RhCE C/T RHDe4S AGACAAACTGGGTATCGTTGC RHD 111 RHDe4AATCTACGTGTTCGCAGCCT Exon 4 RhD/RhCE A/G RHDe9SCCAAACCTTTTAACATTAAATTATGC RHD 98 RHDe9A TTGGTCATCAAAATATTTAGCCTC Exon 9RhC/Rhc T/C RHCEe2S TGTGCAGTGGGCAATCCT RHCE 90 RHCEe2ACCACCATCCCAATACCTG Exon 2 RhE/Rhe C/G RHCEe5S AACCACCCTCTCTGGCCC RHCE107 RHCEe5A ATAGTAGGTGTTGAACATGGCAT Exon 5 GYPBS/GYPBs T/C GYPBe4SACATGTCTTTCTTATTTGGACTTAC GPYB 103 GYPBe4A TTTGTCAAATATTAACATACCTGGTACExon 4 K/k T/C KELe6S TCTCTCTCCTTTAAAGCTTGGA KEL 142 KELe6AAGAGGCAGGATGAGGTCC Exon 6 Kp^(a)/Kp^(b) T/C KELe8S AGCAAGGTGCAAGAACACTKEL 100 KELe8A AGAGCTTGCCCTGTGCCC Exon 8 Fy/Fy0 T/C FYproSTGTCCCTGCCCAGAACCT Duffy 90 FYproA AGACAGAAGGGCTGGGAC PromoterFy^(a)/Fy^(b) G/A FYe2S AGTGCAGAGTCATCCAGCA Duffy 122 FYe2ATTCGAAGATGTATGGAATTCTTC Exon 2 Jk^(a)/Jk^(b) G/A JKe9SCATGAACATTCCTCCCATTG Kidd 130 JKe9A TTTAGTCCTGAGTTCTGACCCC Exon 9Di^(a)/Di^(b) T/C DIe19S ATCCAGATCATCTGCCTGG Diego 90 Die19ACGGCACAGTGAGGATGAG Exon 19 HPA-1a/b T/C GP3Ae3S ATTCTGGGGCACAGTTATCCGP3A 114 GP3Ae3A ATAGTTCTGATTGCTGGACTTCTC Exon 3 The above primerscorrespond to SEQ ID NOs 1-24, respectively, as outlined herein above.

TABLE 1A Additional Blood Group and Platelet Antigen SNPs for ClinicallyRelevant Antigens. Product Antigen SNP Target Size (bp) A/O G/T ABOGalNAc/Del Exon 6 A/B C/G ABO (GalNAc/Gal) Exon 7 A/B G/A ABO(GalNAc/Gal) Exon 7 A/B C/A ABO (GalNAc/Gal) Exon 7 A/B G/C ABO(GalNAc/Gal) Exon 7 M/N G/A MNS Exon 2 M/N T/G MNS Exon 2 MNS/MiI C/TMNS Exon 3 RHD/Weak D T/G RHD Type 1 Exon 6 RHD/Weak D G/C RHD Type 2Exon 9 RHD/Weak D C/G RHD Type 3 Exon 1 RHD/D nt602 C/G RHD VariantsExon 4 RHD/‘DAR’ T/C RHD Variant Exon 7 RHD/Weak D C/A RHD Type 5 Exon 3RHD/D_(el) G/A RHD IVS3 + 1 RHD/D_(el) G/T RHD Exon 6 RHD/D_(el) G/A RHDExon 9 RHD/RHD_(ψ) A/T RHD nt506 Exon 4 RHCE/RhC T/C RHCE IVS2 + 1722RHCE/RhC C/T RHCE IVS2_1751 RHCE/ C/G RHCE VS variant Exon 5Lu^(a)/Lu^(b) A/G LU Exon 3 Au^(a)/Au^(b) A/G LU Exon 12 Js^(a)/Js^(b)C/T KEL Exon 17 Js/Js_(null) G/T JK IVS7 + 1 FY/Fy^(x) C/T FY Exon 2FY/Fy^(x) G/A FY Exon 2 Wr^(a)/Wr^(b) A/G DI Exon 16 Yt^(a)/Yt^(b) C/AYT Exon 2 Sc1/Sc2 G/A SC Exon 3 Do^(a)/Do^(b) C/T DO (nt 378) Exon 2Do^(a)/Do^(b) T/C DO (nt 624) Exon 2 Do^(a)/Do^(b) A/G DO (nt 793) Exon2 Co^(a)/Co^(b) C/T CO Exon 1 In^(a)/In^(b) C/G IN Exon 2 Ok(a+)/Ok(a−)G/A OK Exon 4 GIL/GIL_(null) G/A GIL IVS5 HPA-2a/b C/T GP1BA Exon 2HPA-3a/b T/G GP2B Exon 26 HPA-4a/b G/A GP3A Exon 4 HPA-5a/b G/A GP1AExon 13 Gov^(a)/Gov^(b) A/C CD109 Exon 19

Each antigen listed on the left represents a blood group or HPA genotypeand the single nucleotide polymorphism (SNP). Some genotypes areevaluated using more than one SNP because they differ by more than onenucleotide. Each PCR primer pair consists of a sense (Primer Name endingin S) and antisense (Primer Name ending in A) oligonucleotide (Sequence5′-3′) designed to amplify the DNA region containing the SNP for theantigen of interest. The target region (Product Target) and theamplified fragment (Size (bp)) are shown on the right. Note that 12 SNPsare evaluated for 19 different blood group and platelet antigens becausesome antigens have more than one SNP. In some cases an A or G SNP isincluded since the complementary DNA strand can be evaluated as it willcontain the T or C SNP of interest.

TABLE 2Extension probes used to detect the nucleotides of blood group and HPA SNPs.Name Sequence 5′-3′ RHD GTGATTCTGTACGTGTCGCC GTCTGATCTTTATCCTCCGTTCCCTExon 4 RHD GCGGTAGGTTCCCGACATAT TTTAAACAGGTTTGCTCCTAAATCT Exon 9 RHCEGGATGGCGTTCCGTCCTATT GGACGGCTTCCTGAGCCAGTTCCCT Exon 2 RHCECGACTGTAGGTGCGTAACTCGATGTTCTGGCCAAGTGTCAACTCT Exon 5 GYPBAGGGTCTCTACGCTGACGAT TTGAAATTTTGCTTTATAGGAGAAA Exon 4 KELAGCGATCTGCGAGACCGTAT TGGACTTCCTTAAACTTTAACCGAA Exon 6 KELAGATAGAGTCGATGCCAGCT TTCCTTGTCAATCTCCATCACTTCA Exon 8 FYGACCTGGGTGTCGATACCTA GGCCCTCATTAGTCCTTGGCTCTTA Promoter FY ExonACGCACGTCCACGGTGATTT GGGGGCAGCTGCTTCCAGGTTGGCA 2 JK ExonCGTGCCGCTCGTGATAGAAT AAACCCCAGAGTCCAAAGTAGATGT 9 Di ExonGGCTATGATTCGCAATGCTT GTGCTGTGGGTGGTGAAGTCCACGC 19 GP3AAGAGCGAGTGACGCATAC TTGGGCTCCTGTCTTACAXGCCCTGCCTC Exon 3 The above probescorrespond to SEQ ID NOs 25-36, respectively, as identified hereinabove. The DNA bases are represented by their single letter equivalents(A, C, G or T) and the letter X in GP3A, Fxon 3, between the SEQ ID NO:36 AND SEQ ID NO: 37 represents a C3 (phosphoramidite) spacer betweenthe two adjacent DNA bases.

The present invention also provides novel hybrid probes, wherein thepreferred probes are listed in Table 2, but limited to said listing.Each extension probe is designed in two parts: (1) the 5′ portion: the5′ nucleotides indicated in boldface of the extension primer arecomplementary to unique and specific DNA sequences which aremicro-arrayed onto the bottom of microplates in a specified location ofeach microplate well. Thus, the 5′ portion of the extension probes intable 2 represent, but are not limited to, 12 unique complementarysequences used together to identify the individual SNPs throughhybridization to the micro-arrayed tags in the microplate wells. The 12unique 5′ portions can be interchanged with each of the 3′ regionsspecified below, which contain DNA sequences complementary to andadjacent to the SNPs of interest, or they can be interchanged with otheradditional unique 5′ portions as specified by the micro-arrayed tags inthe microplate wells provided they are used to identify blood group orHPA SNPs; and (2) the 3′ portion: the 3′ nucleotides are complementaryto and precisely adjacent to the SNP site of the PCR-amplified DNA,which enables the detection of either or both nucleotides of the SNP.Thus, the extension probe is a unique sequence that can hybridized to aspecific location and to the PCR-amplified DNA and be extended by asingle fluorescent-labeled dideoxy-nucleotide using PCR thermal cylers.The extension probe products are hybridized to the complementarymicro-arrayed DNA sequence on the microplate and the incorporation ofBodipy- and Tamra-labeled dideoxy-nucleotides are detected bylaser-microplate fluorescence for each individual blood group SNP. Thepresence of the nucleotides for a given SNP is displayed by automatedimaging and analysis software. In one variation of the detectionreaction, a dideoxyguanidine tri-nucleotide labeled with theBodipy-fluorochrome is added in the extension reaction. If adeoxycytidine is present in the PCR-amplified DNA fragment, then thenucleotide will be incorporated into the nascent DNA fragment. Inanother variation of the reaction, a dideoxyadenine nucleotide labeledwith the Tamra-fluorochrome is added to the extension assay. If thePCR-amplified fragment contains a deoxythimidine, then an extension willoccur. In each case, the flurochrome is detected after the extensionreaction has been completed. Again, these reactions proceed in the sametube along with the other extension reactions. The laser-detectionapparatus can identify and evaluate each specified extension due to thelocation of each micro-arrayed DNA sequence.

Each extension primer has a region complementary to a tag that is beenbound to the surface of a microplate well (Bold nucleotides) and aregion (Italicized nucleotides) that is complementary to the region andimmediately adjacent to the SNP site.

It should be noted that the teachings, products and methods of thepresent invention are not limited to the above-specified primer pairsand probes, but additionally comprise all primer pairs and probesspecific to the blood group and HPA SNPs, wherein said primer pairs andprobes are optimized for use in a multiplex PCR reaction for thesimultaneous identification of more than one, or all, blood group or HPAgenotypes and their corresponding phenotypes.

EXAMPLES

Although the following examples may provide preferred methods, products,platforms or protocols of the present invention, it will be understoodby one skilled in the art that the presently provided examples are notlimited to the specified parameters of each example, and may be variedprovided that the resulting outcome of the methods or protocols are inaccordance with the teachings of the present invention, and the productsare functionally equivalent or relating to the teachings of the presentinvention.

Example 1

A preferred protocol for the multiplex blood group and HPA SNPGenotyping is provided. Although the present example analyzes 12 SNPextension primers, the present invention is not limited to the analysisof a maximum of 12 SNPs, but may include a plurality of SNPs relating tomore than one or all of the blood group or HPA SNPs.

Additional blood group and platlet antigen SNPs for clinically relevantantigens embodied by the present invention appear in Table 1A. Primerpairs and probes, such as those exemplified in Tables 1 and 2,corresponding to these SNPs of clinical relevance, can be preparedaccording to the teachings of the present invention. Target primers maybe initially identified from existing databases (e.g. autoprimer.com)based on information corresponding to the SNP of interest and thecorresponding flanking regions, and subsequently optimized as hereindisclosed for use in accordance with the present invention.

I (a). PCR Primer Pooling Step Action 1 Dilute each of 12 PCRS and PCRAprimer (forward and reverse primers) pairs to final concentration of 240uM (only required upon arrival of new primers) 2 Generate working primerpool by combining 5 ul of each of the 24 individual PCR primers

I (b). SNP Extension Primer Pooling Step Action 1 Dilute each of 12 SNPextension primers to final concentration of 120 uM (only required uponarrival of new primers) 2 Generate working SNP extension primer pool bycombining 10 ul of each of the 12 individual SNP extension primers

II. Multiplex PCR from purified DNA templates Step Action 1 Prepare 10ul multiplex PCR master mix for use with 96 well plates containing PCRprimers (synthesized by Integrated DNA Technologies, Coralville, IA,USA), dNTPs (MBI Fermentas, Hanover, MD, USA), MgCl₂, 10X PCR Buffer,and Amplitaq Gold (Applied Biosystems, Branchburg, NJ, USA): InitialFinal Volume Component Concentration Concentration (ul/well) PCR primerpool 10 uM each 50 nM each 0.05 dNTPs 2.5 mM each 75 uM each 0.33 MgCl₂25 mM 5 mM 2.00 10x PCR Buffer 10x 1x 1.00 AmpliTaq Gold 5 U/ul 0.075U/ul 0.15 dH₂O 4.47 2 For each DNA Sample, transfer 2 ul of 4 ng/ulstock DNA to each well of 96 well plates. Use Biomek FX (Beckman CoulterInc., Fullerton, CA, USA) Script ‘2ul96well Transfer’ automated program3 Place Multiplex PCR Master Mix in Biomek FX station 1. Place 96 wellplates of DNA in Biomek FX station 5-8. 4 Transfer 8 ul Multiplex PCRmaster mix to DNA samples using Biomek FX Script: ‘8 ul PCR Transfer’ 5After addition of master mix seal tightly with MJ Microseal A film (MJResearch, Inc., Waltham, MA, USA) 6 Spin down in centrifuge for 30 secat 1500 rpm 7 Place in MJ Tetrad Thermal cyclers (MJ Research, Inc.,Waltham, MA, USA)and run ‘UHT-MPX’ CBS multiplex PCR program: Thermalcycle conditions ‘UHT-MPX’: Denature 94° C. 1:00 (min) 35 cycles of: 94°C. 0:30 (min) 55° C. 0:33 (min) 72° C. 1:00 (min) Hold Temperature  4°C. ∞

III. Post PCR Cleanup Step Action 1 Prepare ExonucleaseI (ExoI; USBCorporation, Cleveland, OH, USA) and Shrimp Alkaline Phosphatase (SAP;USB Corporation, Cleveland, OH, USA) master mix: Component Finalconcentration Volume per well (ul) ExoI 2 U 0.4 SAP 1 U 2.0 10x SAPbuffer 1x 0.6 dH₂O 3.0 2 Add Exo/SAP master mix to grooved reservoir andplace on Multimek (Beckman Coulter Inc., Fullerton, CA, USA) Station 3 3Add UHT (ultra high-throughput) salt solution (provided) to groovedreservoir and place on Multimek Station 4 4 Transfer 8 ul Exo/SAP mastermix to amplified PCR products using Multimek Script: EXO96-2.SCI (two 96well plates, at Multimek stations 1 and 2 5 After Multimek addition ofExo/SAP seal tightly with MJ Microseal A film 6 Spin down in centrifugefor 30 sec at 1500 rpm 7 Place in MJ Tetrad Thermal cyclers and run‘UHTCLEAN’ program: Thermal cycle conditions ‘UHTCLEAN’: Temp Time (min) 37° C. 30:00 100° C. 10:00  4° C. ∞

IV. SNP-IT Assay using the GENOMELAB SNPSTREAM ™ (Beckman Coulter Inc.Fullerton, CA, USA) Step Action 1 Prepare SNP-IT extension mixcontaining extension primers (synthesized by Integrated DNATechnologies, Coralville, IA, USA), C/T ddNTPs, Extension mix diluent,and DNA polymerase (Beckman Coulter Inc., Fullerton, CA, USA) ComponentVolume per well (ul) SNP Extension primer pool  3.22 C/T ddNTP Extensionmix  21.43 Extension mix diluent 402.98 DNA polymerase  2.24 dH2O 318.222 Add SNP-IT mix to grooved reservoir and place on Multimek Station 3 3Add UHT salt solution (provided) to grooved reservoir and place onMultimek Station 4 4 Transfer 7 ul SNP-IT extension mix to UHT-CLEAN PCRproducts using Multimek Script: 7UL96-2.SCI (two 96 well plates, atMultimek stations 1 and 2 5 After Multimek addition of SNP-IT extensionmix seal tightly with MJ Microseal A film 6 Spin down in centrifuge for30 sec at 1500 rpm 7 Place in MJ Tetrad Thermal cyclers and run ‘UHT-SNPIT’ program: Thermal cycle conditions ‘UHTSNPIT’: Temp Time (min)Denature 96° C. 3:00 45 cycles of: 94° C. 0:20 40° C. 0:11 HoldTemperature  4° C. ∞

V. Post-extension Transfer and Hybridization Step Action 1 Preheatincubator to 42° C. 2 Make sure there is adequate 20x dilution ofSNPWare UHT Wash Buffer in washer Carboy B. If required dilute 20x stocksolution with water and refill Carboy B 3 Run SAMI/EL 405 Script ‘PrimeB’ 4 Place all Tag Array plates in Row 1 of the Carousel, starting withHotel 1, with subsequent plates in Hotel 2, 3, etc., preferably withtheir barcodes facing inwards. 5 Place all PCR plates directly belowtheir corresponding Tag Array Plates. PCR plates corresponding toQuadrants 1-4 should be placed in Rows 2-5 of the proper Hotel,respectively. For all PCR plates, the “ABC . . . ” lettered edge of theplates should face inwards on the Carousel. 6 Place grooved reservoirwith solubilized UHT Salt Solution in Multimek Station 4 7 Place groovedreservoir with Hybridization solution master mix in Multimek Station 3Hybridization Solution master mix: Component Volume per Tag Array plate(ul) 2x Hybridiaztion Soluton 3500.00 Hybridization Additive  203.7 8Run SAMI Script ‘Post-extension Transfer_Hybridization 1x384.smt’: Thisautomated program prepares the tag array plate by washing it 3x withSNPWare UHT wash buffer; adds 8.0 ul of Hybridization solution mastermix to each SNP extension reaction and subsequently transfers 8.0 ul ofthis mixture to the prepared tag array plate. 9 Place Tag Array platesin humidified 42° C. incubator for 2 hours

VI. Post-Hybridization Wash Step Action 1 Make sure there is adequate64x dilution of SNPWare UHT Stringent Wash Solution in washer Carboy C.If required dilute 64x stock solution with water and refill Carboy C 2Run SAMI/EL 405 Script ‘Prime C’ 3 Run SAMI/EL 405 Script ‘Post-hyb 3xWash’ 4 Completely dry Tag Array plates using vacuum/pipette tip 5 RunSAMI/EL405 script ‘Prime A’ several times to clean plate washer pins

VII. UHT (Ultra high through-put) Tag Array Plate Reading Step Action 1Turn on lasers, turning both keys 90 degrees clockwise, and allow atleast 30 minutes to warm up 2 Turn on SNPScope Reader and Twister. 3Activate lasers: Flip two switches on laser box from ‘Standby’ to‘Operate’/‘Laser’ 4 Open UHT Run Manager Software and ‘Initialize’SNPScope system 5 Stack Tag Array plates in Twister carousel 1, with‘Assay Test Plate’ on top. Make sure all barcodes are facing outwards,and plates are pushed towards the reader 6 Select ‘SNPTEST_W_BC_run’from UHT RUN Manager Software, enter the number of plates to be read(including the test plate). 7 Select ‘RUN’

The SNPScope plate reader will excite and capture images ofBodipy-fluorescein and Tamra-labeled ddNTPs separately. All genotypecalls are subsequently automatically generated using the SNPStreamSoftware Suite of MegaImage, UHTGetGenos and QCReview.

It should be noted that the specific steps associated with the protocolexemplified in Example 1 are not intended to limit the teachings andmethods of the present invention to the specific above protocol. Example1 is provided to specify a preferred method in accordance with thepresent invention wherein a plurality of blood group and HPA SNPs aresimultaneously analysed in a ultra high throughput multiplex automatedsystem for the determination of the specific genotypes and accordinglythe phenotypes associated therewith. Accordingly, it should beunderstood by one skilled in the art that the steps of Example 1 may bevaried provided that such variations yield the preferred results of thepresent invention.

Results 1. GP3A Exon 3 SNP Scatter Plots.

The robotic UHT platform produces laser-fluorescence values for eachsample which are represented in ‘scatter plots’ for the operator toreview. A sample scatter plot is shown in FIG. 1 for the SNP analysisGP3A Exon 3, which represents the HPA-1a and HPA-1b antigens. As canbeen seen in FIG. 1 and FIG. 4, results are graphed using logarithmicand XY scatter plots (upper right). Green O, orange □ or blue O sampledesignations represent CC, TC and TT SNP genotype calls, respectively,with corresponding graphical summaries appearing in the respectivelegends of each figure. No fluorescence represents an assay failure (FL)for that sample.

Scatter plots (as shown in FIG. 1 and FIG. 4) are generated preferablyusing SNPStream software suite and viewed through QCReview. It should beadditionally noted that the present analysis is not limited to SNPstreamor QCReview, and may be carried out using any SNP analysis software.Individual TT, TC and CC genotype calls are represented as dark blue,orange and green open circles, respectively. Sample failures and watercontrols are represented by yellow and light blue filled circlesrespectively. Logarithmic (left) and XY scatter (upper right) plots aregenerated using the relative fluorescence of the Bodipy-fluorescein andTamra labels obtained during SNPScope plate imaging and analysis.

2. SNP Data Manipulation and Analysis.

The SNP results of a scatter plot are electronically exported to aspreadsheet and examined for total sample failure and individual SNPfailure rates. SNP results for 372 DNA samples are summarized in Table 3(provided in Appendix A). Accordingly, Table 3 provides the Pass andFailure Rates for 12 blood group and HPA SNP analyses. 372 DNA sampleswere analyzed for several antigens, including the blood group RhD (RHDExon 4 and RHD Exon 9) and platelet HPA-1a/b (GP3A Exon 3) genotypes.Sample success or pass rates are indicated on the right and individualSNP success or pass rates are shown at the bottom. Three hundred andfifty seven of 372 samples (96%) had results for at least one SNP.Individual SNP results (i.e. minus the sample failures) ranged from80-100%; only two SNPs had success rates <98%. Individual SNP failuresdo not affect the results of a sample for other SNPs that do not fail.

3. SNP Allele Result Compared to the Serological Result

RhD status was compared between the serological result and the SNPanalysis for RHD Exon 4 and RHD Exon 9. Table 4 summarizes thecomparison. 287 of 291 (98.6%) RhD positive units and 55 of 66 (83.3%)RhD negative units were identified correctly using the UHT SNP platform.It is important to note that the 6 incorrect calls suggesting thepresence of the RHD gene in a serologically RhD-negative sample may bedue to one of the non-functional RHD genes present in the randompopulation (Singleton B. K. et al., Blood 2000; 95:12; Okuda H., et al.,J Clin Invest 1997; 100:373; Wagner F. F. et al., BMC Genet 2001; 2:10).

TABLE 4 A comparison of the SNP genotype result and the serologicalresult obtained with government-regulated antisera. D- RHD Exon RHD Exonpositive: Assay 4 9 No Percent N = 291 pos Pos 287 98.6% neg Neg 4 1.4%Total 291 D- negative: Assay RHD4 RHD9 No Percent N = 66 neg Neg 5583.3% neg FL 5 7.6% pos Pos 6 9.1% Total 66 NOTE: CBS laboratoryregulations do not allow copies of serological results of blood donorsto be made from their laboratory information system. Therefore, theresults of the CBS serological phenotypes were reviewed by researchpersonnel and the results tabulated and compared to the SNP data.

4. SNP Genotype Frequency Analysis.

The SNP results then were compared with published phenotype frequenciesfor Caucasians and Blacks and are summarized in Table 5 below. The dataclearly shows that the allele frequencies are consistent with theaccepted published frequencies for Caucasians and Blacks. The data showthat the SNP genotype frequencies match the published populationphenotype frequencies.

TABLE 5 Table 5. A summary of the UHT SNP analysis of genotypefrequencies for several SNPs analyzed and compared to publishedphenotype frequencies for Caucasians and Blacks. The ethnicity of thesamples analyzed is not known. UHT Genotyping Analysis FL = assayfailure Phenotype Caucasians Blacks Observed (%) K− k+   91%   98% 32691.3 KEL Exon6 K− k+   91%   98% 326 91.3 K+ k−  0.2% rare 0 0 K+ k+ 8.8%   2% 28 7.8 Fails 3 0.8 No of FL  18 No. of Pass 354 Call Rate95.2% An independent assay as described in Molecular Protocols in Trans-fusion Medicine was performed using the UHT SNP Stream System. Sevensamples were tested (Four KEL 2/KEL 2, Three KEL1/KEL 2). All samplesshowed a 100% correspondence with the UHT genotype results. KEL Exon8Kp(a+ b−) Rare   0% 0 0 Kp(a− b+) 97.7%  100% 354 99.2 Kp(a+ b+)  2.3%rare 1 0.3 Fails 2 0.6 No of FL  17 No. of Pass 355 Call Rate 95.4% DIExon18 Di(a+ b−) <0.01%  <0.01%  0 0 Di(a− b+) >99.9%  >99.9%  353 98.9Di(a+ b+) <0.1% <0.1% 2 0.6 Fails 2 0.6 No of FL  17 No. of Pass 355Call Rate 95.4% FY PRM wt/wt 348 97.5 wt/mut 7 20 mut/mut 2 0.5 Fails 00 No of FL 15 No. of Pass 357 Call Rate 96.0% An independent assay asdescribed in Molecular Protocols in Trans- fusion Medicine was performedusing the UHT SNP Stream System. Thirteen samples were tested (sixwt/wt, five wt/mut and two mut/mut for the GATA site). All samplesshowed a 100% correspondence with the UHT genotype results. FY Exon 2Fy(a+ b−)   17%   9% 89 24.9 Fy(a− b+)   34%   22% 112 31.4 Fy(a+ b+)  49%   1% 155 43.4 Fails 1 0.3 No of FL  16 No. of Pass 356 Call Rate95.7% An independent assay as described in Molecular Protocols in Trans-fusion Medicine was performed using the UHT SNP Stream System Elevensamples were tested (eight FY2/FY2, three FY1/FY2 and one FY1/FY1). Allsamples showed a 100% correspondence with the UHT genotype results. GP3AExon 3 HPA-1a/1a   80%   84% 263 73.7 HPA-1a/1b   18%   64% 89 24.9HPA-1b/1b   2%   0% 4 1.1 Fails 1 0.3 No of FL  16 No. of Pass 356 CallRate 95.7% An independent assay as described in Molecular Protocols inTrans- fusion Medicine was performed using the UHT SNP Stream System.Eighteen samples were tested (Seven HPA-1a, Seven HPA-1a/1b and FourHPA-1b). All samples showed a 100% correspondence with the UHT genotyperesults. JK9 Jk(a+ b−) 26.3% 51.1% 90 25.2 Jk(a− b+) 23.4%  8.1% 87 24.4Jk(a+ b+) 50.3% 40.8% 178 49.4 Fails 2 0.5 No of FL  17 No. of Pass 355Call Rate 95.4% An independent assay as described in Molecular Protocolsin Trans- fusion Medicine was performed using the UHT SNP Stream System.Nineteen samples were tested (Seven JK1, Seven JK1/JK2 and Five JK2).All samples showed a 100% correspondence with the UHT genotype results.5. HPA-1a/HPA-1b PCR-RFLP Analysis.

The GP3A Exon 3 SNP detection method for HPA-1a/b genotyping (AppendixA) was compared to a subset of samples (n=18) using conventionalPCR-RFLP analysis performed independently (FIG. 2). The results of thetwo assays were 100% concordant. In addition, a 217G nucleotide mutation21 basepairs downstream of the GP3A SNP was present in sample 8. Thismutation does not affect HPA-1b expression but is detected in thePCR-RFLP and is prone to interpretation error in the conventionalPCR-RFLP assay. However, the sample was correctly genotyped as HPA-1b inour SNP assay. Accordingly, the present invention eliminates orminimizes error in HPA-1 results obtained since no confusing orconfounding information results from the gel readings of the presentinvention. That is to say, the conventional RFLP detected the presenceof an additional DNA fragment at ˜180 bp which represents a heterozygousHPA-1b/1b^(G217) allele and was correctly genotyped as HPA-1b/b by thepresent invention.

Example 2

However, it should be obvious to one skilled in the art that othermethodologies and/or technologies for SNP identification could be used,providing that the novel DNA sequences disclosed above are also used.Other embodiments could include the following but without limitation tomicro-arrays on glass slides or silica chips, the use of massspectrometry, or oligo-ligation and extension techniques to detect theSNPs of interest.

A preferred method of the present invention relates to a method for thedetection of blood group and HPA genotypes. The present invention alsoprovides novel DNA sequences that are used as primers in a multiplex PCRformat according to the present invention to amplify the genomic regionsof interest. The present invention also provides novel combinations ofDNA sequences that are used in said multiplex PCR format, and for novelDNA sequences that are used to detect blood group and platelet SNPs.

A preferred application of the present invention is in the bloodcollection and blood banking industry without limitation to red bloodcell, platelet, and bone marrow donations. Canada has over 850,000 blooddonations yearly, many from repeat donors. Eventually, after all repeatdonors are tested (each donor is tested once), the analyses will beperformed only on the blood of new donors. With over 29 blood group and6 HPA systems encompassing over 250 antigens, the platform will findwide application in this industry.

The present invention additionally encompasses various embodimentsrelating to the detection of various SNPs for the determination of thevarious genotypes in a sample and for the determination of thecorresponding phenotype. In a preferred embodiment, the presentinvention utilizes a platform to analyzes a cytidine-to-thymidine (C→T)single nucleotide polymorphism. The invention may also employ themultiplex detection of, but not limited to, C→A, A→T, and G→C SNPs, orany other nucleotide SNP related to blood group or platelet antigens.

The present invention may additionally include methods and products forthe detection of clinically relevant blood group antigens whereby anantigen of interest is characterized by a genotypic identifier thatexceeds a single nucleotide polymorphism. Specifically, the presentinvention may extend to include clinically relevant insertions ordeletions or other nucleotide changes that characterize a blood groupantigen of interest, such a multiple base pair insertion in an allele ofinterest. For example, a genotypic identifier corresponding to a bloodgroup antigen of interest may be pre-characterized, suitable primers andprobes for detection thereof may be prepared and a blood sample screenedaccording to the teachings of the present invention.

The present invention provides DNA sequences corresponding to the PCRprimer pairs optimized for multiplex use to identify blood group andplatelet antigens simultaneously. Accordingly, the present inventionprovides the novel primer pair sequences listed in Table 1.

The present invention additionally provides novel DNA sequences used toidentify the single nucleotide polymorphisms (SNPs) that representunderlying DNA blood group and platelet antigens. Accordingly, thepresent invention provides the novel extension probes listed in Table 2.

The present invention provides a method of a combined analysis of bloodgroup and HPA SNP analyses.

The present invention advantageously utilizes PCR, the variant andunique SNPs for the variant alleles that infer blood group phenotypes,and single base extension and detection chemistry as a foundation forthe novel products and methods of the present invention. Accordingly,the present invention provides a high throughput, multiplexed, DNA-basedmethod of blood group genotyping that replaces the current manual,semi-automated and automated serological screening process used todetermine blood group phenotypes.

Accordingly, the present invention provides a method for theidentification of rare blood group genotypes due to the suite of SNPs asdescribed above, and in some instances replaces the current state of theart in which most rare blood group genotypes are identifiedserendipitously (propositus and their relatives) and enablingsignificant advances over current serological technologies. For example,by analyzing the SNP for the RhC allele in Rh negative blood, we canidentify RhC homozygotes and thereby, the rare RhD-negative andRhc-negative blood.

The present invention additionally provides a method of use in tissuecompatibility matching for the purposes, without limitation, of organtransplantation, bone marrow transplantation and blood transfusionrelated to blood group and platelet antigens.

The present invention additionally provides novel components andconstituents that are beneficial for the analyses relating to thepresent invention. More specifically, the group of currently developedSNPs representing a ‘suite’, or the presently known set of SNPs thatrelate to clinically important blood group and HPA genotypes for redblood cell and platelet antigens, respectively are provided. The presentinvention is not limited to the presently listed SNPs, but is understoodto comprise all blood group and platelet antigen, and preferably HPASNPs that may be analyzed in accordance with the teachings of thepresent invention and using the products, protocols and methods of thepresent invention.

The present invention also provides the DNA primer sequences optimizedfor use in a multiplex PCR format.

The present invention also provides novel DNA probe sequences used todetect the SNPs of interest.

The present invention provides a method for the simultaneous detectionof a plurality of blood group SNPs. More specifically, the presentinvention provides a method for the simultaneous detection of at least19 blood group SNPs; RHD (2), RHC/c, RHE/e, S/s, Duffy (a/b), Kidd(a/b), Diego (a/b), Kell K1/K2, Kell K3/K4, and HPA-1a/b simultaneously.The method of the present invention provides (1) DNA sequencescorresponding to the PCR primer pairs optimized for multiplex use toidentify a plurality of blood group and platelet antigenssimultaneously; (2) Novel DNA sequences used to identify the singlenucleotide polymorphisms (SNPs) that represent underlying DNA bloodgroup and platelet antigens; and (3) The combination of SNP analysesincluding blood group and platelet antigens.

To support and validate the teachings of the present invention variousexperimental tests have been completed and analyzed. Numerous validatingexperimental data has been recorded, however, for the purpose ofsimplicity the following example is provided. Each step in thevalidating experiment is noted below:

(1) Ultra high throughput (UHT) Multiplex SNP analyses on 372 unrelatedblood donor specimens for RHD (2), RHC/c, RHE/e, S/s, FY1/FY2 (2),JK1/JK2, DI1/DI2, KEL1/KEL2, KEL3/KEL4, and HPA-1A/B genotypes andcorresponding phenotypes was examined, and data was recorded (pleaserefer to Appendix A for the raw data accumulated, and Table 5 for aSummary of the results obtained).

(2) Manual PCR-RFLP analyses was performed on some of the 372 specimensfor some of the blood group SNPs to for comparison to the resultsobtained in Step (1).

(3) Serological analyses was also performed on some of the 372 specimensfor each of the blood group and HPA SNPs using Health Canada regulatedreagents performed by licensed medical technologists in a provinciallylicensed laboratory.

(4) Serological analyses was also performed on some of the 372 specimensfor each of the blood group and platelet antigens by unlicensed researchtechnologists using Health Canada regulated reagents and methodologiesin an unlicensed laboratory.

The results obtained from the above validating experimental data isprovided below by way of supportive Figures and Tables.

1. SNP Platform Data Generation.

The robotic platform produces fluorescence for each sample which arepresented in ‘scatter plots’ (as illustrated in FIG. 1) for the operatorto review. Sample genotype results are shown for each blood group SNPand are graphed using logarithmic and XY scatter plots (upper right).Green, orange or blue sample designations represent CC, TC and TTgenotype calls respectively. No fluorescence represents an assay failure(FL) for that sample.

2. SNP Data Manipulation and Analysis.

The SNP results of a scatter plot are electronically exported to aspreadsheet and examined for total sample failure and individual SNPfailure rates. Twelve SNP results for 372 DNA samples are summarized inTable 3 with sample failure rates (shown on the right) and individualSNP success rates (shown at the bottom). Three hundred and fifty sevenof 372 samples (96%) had results for at least one SNP. Individual SNPresults ranged from 80% to 100%; only one SNP result success rate was<98%. Individual SNP failures do not affect the results of a sample forother SNPs that do not fail.

3. SNP Allele Frequency Analysis.

The SNP results where then compared with published phenotype frequenciesfor Caucasians and Blacks and are summarized in Table 5 above. The datashows that the allele frequencies are consistent with the acceptedpublished frequencies for Caucasians and Blacks.

3.1 SNP Allele Result Compared to the Serological Result.

RhD status was compared between the serological result and the SNPanalysis for RHD exon 4, and 9 (RHD Exon 4, RHD Exon 9, respectively).Table 4 summarizes the comparison. 287 of 291 (98.6%) RhD positive unitsand 55 of 66 (83.3%) RhD negative units were identified correctly usingthe UHT SNP platform.

3.2 SNP Analysis Compared to Manual PCR-RFLP.

Some of the UHT SNP genotype results were compared with manual PCR-RFLPanalysis performed independently. The results show 100% concordance. Arepresentative PCR-RFLP is shown in FIG. 3.

The genotyping technology provided in the present invention queries andanalyzes SNPs using single base-pair primer extension. In brief, thegenomic region surrounding the SNP of interest is amplified and used asa template for the ensuing hybridization and single nucleotide extensionof the SNP specific extension primer. The extension primer is designedto hybridize adjacent to the polymorphic nucleotide(s) and enables us toquery bi-allelic polymorphisms, small insertions, deletions orinversions. The 5′ extension primer tags are hybridized to thecomplementary DNA sequence on micro-arrayed plates and incorporation ofBidopy- and Tamra-labeled ddNTPs are detected by laser-microplatefluorescence for each individual blood group and HPA SNP. Individualsample genotypes are generated through automated imaging and analysissoftware as shown in the genotype scatter plots of FIG. 1.

The embodiment(s) of the invention described above is(are) intended tobe exemplary only. The scope of the invention is therefore intended tobe limited solely by the scope of the appended claims.

APPENDIX A Genotype Results for updated 12 SNP CBS Panel Sample SamplePass ID RHD4 RHD7 RHD9 RHCE2 RHCE5 KEL6 KEL8 DI18 FYP FY2 GP3A JK9 FLRate BB24401 FL FL FL FL FL FL FL FL FL FL FL FL 12 0.0% BB24402 TT FLCC CC TC CC CC CC TT CC TC CC 1 91.7% BB24407 TC TT TC TC TC CC CC CC TTTT TC TC 0 100.0% BB24408 TC TT TC TC CC CC CC CC TT CC TT TC 0 100.0%BB24409 TC TT TC TC TC CC CC CC TT CC CC TC 0 100.0% BB24410 TC TT TC TCTC CC CC CC TT TC TT TC 0 100.0% BB24415 TC TT TC TC FL CC CC CC TT TTTT TC 1 91.7% BB24416 FL FL FL FL FL FL FL FL FL FL FL FL 12 0.0%BB24417 TC TT TC FL TC CC CC CC TT CC TT TC 1 91.7% BB24420 TC TT TC TCCC CC CC CC TT CC TT CC 0 100.0% BB24421 TC TT TC TC CC CC CC CC TT TCTT TT 0 100.0% BB24422 TC TT TC FL TC CC CC CC TT TC TC TC 1 91.7%BB24423 TC TT TC TC TC TC CC CC TT TC TT TT 0 100.0% BB24424 TC TT TC FLTC CC CC CC TT CC TT TC 1 91.7% BB24425 TC TT TC TC CC CC CC CC TT TT TTTC 0 100.0% BB24426 TC TT TC TC TC CC CC CC TT TC TT TC 0 100.0% BB24427TC TT TC TC TC TC CC CC TT TC TC CC 0 100.0% BB24428 TC TT TC TC CC CCCC CC TT TT TT TC 0 100.0% BB24429 TC TT TC TC CC CC CC CC TT TC TT TC 0100.0% BB24430 TC TT TC TC TC TC CC CC TT TT TC CC 0 100.0% BB24431 TCTT TC TC CC TC CC CC TT TT TC TT 0 100.0% BB24432 TC TT TC TC CC CC CCCC TT TT TC TT 0 100.0% BB24433 TC TT TC TC CC CC CC CC TT TC TT TC 0100.0% BB24434 TC TT TC TC CC CC CC CC TT CC TT TT 0 100.0% BB24435 TCTT TC TC CC CC CC CC TT TC TT TC 0 100.0% BB24436 TT FL CC CC TC CC CCCC TT TC TT TC 1 91.7% BB24437 TC TT TC TC TC CC CC CC TT TT TT TT 0100.0% BB24438 TC TT TC TC TC CC CC CC TT TT TT TT 0 100.0% BB24439 TCTT TC TC CC CC CC CC TT TC TC TC 0 100.0% BB24440 TC TT TC FL TC CC CCCC TT CC TT TC 1 91.7% BB24444 TC TT TC TC CC TC CC CC TT TC TT TC 0100.0% BB24448 TT FL CC CC FL CC CC CC TT TC TT CC 2 83.3% BB24461 TC TTTC TC CC CC CC CC TT TC TC CC 0 100.0% BB24462 TT FL CC CC TC CC CC CCTT TC TT CC 1 91.7% BB24463 TC TT TC TC CC CC CC CC TT TT TT TC 0 100.0%BB24464 TC TT TC TC CC CC CC CC TT TC TC TC 0 100.0% BB24465 TC TT TC TCTC CC CC CC TT TT TT TC 0 100.0% BB24466 TC TT TC FL TC CC CC CC TT TTTC TC 1 91.7% BB24467 TT FL CC CC TC CC CC CC TT TC TC TC 1 91.7%BB24468 TC TT TC FL TC CC CC CC TT CC TT TC 1 91.7% BB24469 TC TT TC TCCC CC CC CC TT TC TT TC 0 100.0% BB24470 TT FL CC CC TC CC CC CC TT TTCC CC 1 91.7% BB24471 TC TT TC TC TC CC CC CC TT TT TT TC 0 100.0%BB24472 TC TT TC TC TC CC CC CC TT TC TC CC 0 100.0% BB24473 TC TT TC TCTC TC CC CC TT TT TT CC 0 100.0% BB24474 TC TT TC TC TC CC CC CC TT CCTT TT 0 100.0% BB24475 TC TT TC TC TC CC CC CC TT TC TT TC 0 100.0%BB24476 TC TT TC TC CC CC CC CC TT TT TT TC 0 100.0% BB24477 TC TT TC TCTC CC CC CC TT CC TT TC 0 100.0% BB24478 TC TT TC TC TC CC CC CC TT TTTT TC 0 100.0% BB24479 TC TT TC TC TC CC CC CC TT TT TT TC 0 100.0%BB24480 TC TT TC TC TC CC CC CC TT TC TT CC 0 100.0% BB24481 TC TT TC TCCC CC CC CC TT CC TT TT 0 100.0% BB24482 TC TT TC TC TC CC CC CC TT TCTT TC 0 100.0% BB24483 TT FL CC CC TC CC CC CC TT TT TT TC 1 91.7%BB24484 TC TT TC TC TC TC CC CC TT CC TC TC 0 100.0% BB24485 TT FL CC FLTC TC CC CC TT TC TC TC 2 83.3% BB24486 TT FL CC CC TC CC CC CC TT TT TTTT 1 91.7% BB24487 TC TT TC TC TC CC CC CC TT TT TC CC 0 100.0% BB24488TC TT TC TC TC CC CC CC TT CC TT TC 0 100.0% BB24489 TC TT TC TC TC CCCC CC TT TT TC CC 0 100.0% BB24491 TC TT TC TC TC CC CC CC TT TT TT TC 0100.0% BB24492 TT FL CC CC TC CC CC CC TT CC TT CC 1 91.7% BB24493 TT FLCC CC TC CC CC CC TT TC TT TC 1 91.7% BB24494 FL FL FL FL FL FL FL FL FLFL FL FL 12 0.0% BB24495 TC TT TC TC TC CC CC CC TT TC TC TC 0 100.0%BB24496 TC TT TC TC TC CC CC CC TT TT TT CC 0 100.0% BB24497 TC TT TC TCCC CC CC CC TT TC TC TC 0 100.0% BB24499 TC TT TC TC TC CC CC CC TT TTTT CC 0 100.0% BB24504 TC TT TC TC TC CC CC CC TT TT TC TC 0 100.0%BB24505 TC TT TC TC TC CC CC CC TT TC TT TC 0 100.0% BB24506 TC TT TC TCCC CC CC CC TT TT TC TC 0 100.0% BB24507 TC TT TC TC CC CC CC CC TT TCTT TC 0 100.0% BB24512 TT FL CC CC TC CC CC CC TT TC TC TC 1 91.7%BB24513 TC TT TC FL TC CC CC CC TT CC TT CC 1 91.7% BB24516 TC TT TC TCCC CC CC CC TT TT TC TT 0 100.0% BB24517 TT FL CC CC TC TC CC CC TT TCTT TT 1 91.7% BB24518 TC TT TC TC TC CC CC CC TT CC TC TC 0 100.0%BB24519 TC TT TC TC CC CC CC CC TT TC TC CC 0 100.0% BB24522 TC TT TC TCCC CC CC CC TT TC TT TT 0 100.0% BB24523 FL FL FL FL FL FL FL FL FL FLFL FL 12 0.0% BB24524 TC TT TC TC CC CC CC CC TT CC TT TC 0 100.0%BB24525 TC TT TC FL TC CC CC CC TT TC TT TT 1 91.7% BB24526 TC TT TC TCCC CC CC CC TT TC TT TC 0 100.0% BB24527 TT FL CC CC TC CC CC CC TT CCTT TC 1 91.7% BB24528 TC TT TC TC TC CC CC CC TT CC TT TC 0 100.0%BB24529 TC TT TC TC TC CC CC CC TT TC TT TC 0 100.0% BB24530 TC TT TC TCCC CC CC CC TT TC TC CC 0 100.0% BB24531 FL FL FL FL FL FL FL FL FL FLFL FL 12 0.0% BB24532 TC TT TC TC CC CC CC CC TT TT TT TC 0 100.0%BB24533 TC TT TC TC TC CC CC CC TT CC TT CC 0 100.0% BB24534 TC TT TC FLTC TC CC CC TT TT TT TT 1 91.7% BB24535 TC TT TC TC TC TC CC CC TT TC TTTC 0 100.0% BB24536 TC TT TC TC TC CC CC CC TT TC TT TT 0 100.0% BB24537TC TT TC TC CC CC CC CC TT TT TT TC 0 100.0% BB24538 TC TT TC TC CC CCCC CC TT TT TT TC 0 100.0% BB24539 TC TT TC TC CC CC CC CC TT TC TT TT 0100.0% BB24540 TC TT TC TC TC CC CC CC TT TC TT TC 0 100.0% BB24541 TCTT TC FL TC CC CC CC TT TT TT TC 1 91.7% BB24542 TC TT TC TC CC CC CC CCTT TC TT CC 0 100.0% BB24543 TC TT TC TC CC CC CC CC TT CC TT TT 0100.0% BB24547 FL FL FL FL FL FL FL FL FL FL FL FL 12 0.0% BB24548 TT FLCC CC TC CC CC CC TT CC TT TC 1 91.7% BB24549 TT FL CC CC FL CC CC CC TTTC TT TC 2 83.3% BB24550 TC TT TC TC TC CC CC CC TT TC TT CC 0 100.0%BB24552 TC TT TC TC TC CC CC CC TT TC TT CC 0 100.0% BB24553 TC TT TC TCCC CC CC CC TT CC TT TT 0 100.0% BB24554 TC TT TC FL TC CC CC CC TT TTTT TC 1 91.7% BB24555 TC TT TC TC TC CC CC CC TT TT TT TT 0 100.0%BB24556 TC TT TC TC CC CC CC CC TT TT TT TT 0 100.0% BB24557 TC TT TC TCCC CC CC CC TT TT TT TC 0 100.0% BB24558 TC TT TC TC TC CC CC CC TT CCTT TC 0 100.0% BB24559 TC TT TC TC TC CC CC CC TT CC TT TC 0 100.0%BB24560 TT FL CC CC CC CC CC CC TT CC TT CC 1 91.7% BB24561 TC TT TC FLTC CC CC CC TT CC TC TC 1 91.7% BB24562 TC TT TC TC CC CC CC CC TT CC TTTC 0 100.0% BB24563 TT FL CC CC TC CC CC CC TT TT TC TC 1 91.7% BB24564TC TT TC TC CC CC CC CC TT TC TT TC 0 100.0% BB24565 TC TT TC TC TC CCCC CC TT TT TC TC 0 100.0% BB24566 TT FL CC CC TC CC CC CC TT TC TT TC 191.7% BB24567 TC TT TC TC CC CC CC CC TT TC TT CC 0 100.0% BB24568 TC TTTC TC CC CC CC CC TT TC TT TT 0 100.0% BB24569 TC TT TC TC TC CC CC CCTT CC TT CC 0 100.0% BB24570 TC TT TC FL TC CC CC CC TT TT TC CC 1 91.7%BB24571 TC TT TC TC TC CC CC CC TT CC TT TT 0 100.0% BB24572 TT FL CC CCTC TC CC CC TT TC TT TC 1 91.7% BB24573 TC TT TC TC TC CC CC CC TT TC TTCC 0 100.0% BB24574 TT FL CC CC TC CC CC CC TT TC TT CC 1 91.7% BB24575TT FL CC CC TC CC CC CC TT TC TT CC 1 91.7% BB24576 TC TT TC TC TC CC CCCC TT TT TT TC 0 100.0% BB24577 TC TT TC TC TC TC CC CC TT TT TT CC 0100.0% BB24578 TC TT TC TC TC CC CC CC TT TC TC TC 0 100.0% BB24579 TCTT TC TC TC CC CC CC TT CC TT CC 0 100.0% BB24580 TT FL FL CC TC CC CCCC TT TC TT CC 2 83.3% BB24581 TC TT TC TC TC CC CC CC TT CC TT CC 0100.0% BB24586 TC TT TC TC TC CC CC CC TT CC TT TC 0 100.0% BB24587 TCTT TC TC CC CC CC CC TT TC TT CC 0 100.0% BB24594 TC TT TC TC TC CC CCCC TT TC TT TC 0 100.0% BB24600 TC TT TC FL TC CC CC CC TT TT TT TC 191.7% BB24601 TC TT TC FL TC CC CC CC TC TC TT CC 1 91.7% BB24602 TC TTTC TC CC CC CC CC TT TC TT TC 0 100.0% BB24603 TC TT TC FL TC CC CC CCTT TC TT TT 1 91.7% BB24604 TC TT TC TC TC CC CC CC TT TC TC TC 0 100.0%BB24605 TC TT TC TC TC CC CC CC TT TT TC TC 0 100.0% BB24606 TC TT TC TCCC CC CC CC TT TT TT CC 0 100.0% BB24607 TC TT TC TC TC CC CC CC TC TTTC CC 0 100.0% BB24608 TC TT TC TC CC CC CC CC TT TT TT CC 0 100.0%BB24609 TC TT TC FL TC CC CC CC TT CC TC CC 1 91.7% BB24610 TT FL CC CCTC CC CC CC TT TC TT TT 1 91.7% BB24611 TC TT TC TC CC CC CC CC TT CC TTTT 0 100.0% BB24612 TC TT TC TC CC CC CC CC TT CC TT TC 0 100.0% BB24613TC TT TC TC TC CC CC CC TT CC TT TT 0 100.0% BB24614 TC TT TC TC CC CCCC CC TT TT TT TT 0 100.0% BB24615 TC TT TC TC CC TC CC CC TT TT TT CC 0100.0% BB24616 TC TT TC TC TC TC CC CC TT TC TT TT 0 100.0% BB24617 TCTT TC FL TC CC CC CC TT TC TT TT 1 91.7% BB24618 TC TT TC TC TC CC CC TCTT TC TC TC 0 100.0% BB24619 TC TT TC FL FL CC CC CC TT TT TT TC 2 83.3%BB24620 TC TT TC FL FL CC CC CC TT CC TT TC 2 83.3% BB24621 TC TT TC TCTC CC CC CC TT TC TT CC 0 100.0% BB24622 TC TT TC FL TC CC CC CC TT CCTT TC 1 91.7% BB24623 TT FL CC CC TC CC CC CC TT TC TT CC 1 91.7%BB24624 TT FL CC CC TC CC CC CC TT TT TC TC 1 91.7% BB24625 TC TT TC TCTC CC CC CC TT TT TT TT 0 100.0% BB24626 TC TT TC TC CC CC CC CC TT TCTC TC 0 100.0% BB24627 TC TT TC TC TC CC CC CC TT CC TT TT 0 100.0%BB24628 TC TT TC TC TC TC CC CC TT TC TC TC 0 100.0% BB24629 FL FL FL FLFL FL FL FL FL FL FL FL 12 0.0% BB24630 TC TT TC TC CC CC CC CC TT TT TTTT 0 100.0% BB24631 TC TT TC FL TC CC CC CC TT CC TT TC 1 91.7% BB24632TC TT TC TC TC CC CC CC TC TT TT TC 0 100.0% BB24633 TC TT TC FL TC CCCC CC TT TT TC TC 1 91.7% BB24634 TC TT TC TC TC CC CC CC TT CC TT TC 0100.0% BB24635 TC TT TC TC TC CC CC CC TT CC TT CC 0 100.0% BB24636 TCTT TC FL TC CC CC CC TT CC TT CC 1 91.7% BB24637 TC TT TC FL TC CC CC CCTT TT TT TT 1 91.7% BB24638 TC TT TC TC TC CC CC CC TT CC TT TC 0 100.0%BB24639 TT FL CC CC TC CC CC CC TT CC TT TC 1 91.7% BB24640 TC TT TC TCCC CC CC CC TT TT TT CC 0 100.0% BB24641 TT FL CC CC TC CC CC CC TT TTTT TT 1 91.7% BB24642 TC TT TC TC CC CC CC CC TT TC TT CC 0 100.0%BB24643 TC TT TC TC CC CC CC CC TT TC TT TT 0 100.0% BB24644 TT FL FL FLTC FL CC CC TT TC TT CC 4 66.7% BB24645 TC TT TC TC CC CC CC CC TT TT TTCC 0 100.0% BB24646 TT FL CC CC TC CC CC CC TT TT TT TT 1 91.7% BB24647TC TT TC TC TC TC CC CC TT TC TT TC 0 100.0% BB24648 TC TT TC TC TC CCCC CC TT CC TT TC 0 100.0% BB24649 TC TT TC TC TC CC CC CC TT TT TC TT 0100.0% BB24650 FL FL FL FL FL FL FL FL FL FL FL FL 12 0.0% BB24651 TC TTTC TC TC CC CC CC TT TC TT TC 0 100.0% BB24652 TC TT TC TC TC CC CC CCTT TC TT TT 0 100.0% BB24653 TC TT TC TC TC CC CC CC TT CC TC TT 0100.0% BB24654 TC TT TC TC TC CC CC CC TT CC TT CC 0 100.0% BB24655 TTFL CC CC TC CC CC CC TT TC TC TC 1 91.7% BB24656 TT FL CC CC TC CC CC CCTT TT TC TC 1 91.7% BB24657 FL FL FL FL FL FL FL FL FL FL FL FL 12 0.0%BB24658 TT FL CC CC TC CC CC CC TT CC TT TC 1 91.7% BB24659 TC TT TC TCTC CC CC CC TT TC TC TC 0 100.0% BB24660 TT FL CC CC TC CC CC CC TT TTTT TC 1 91.7% BB24661 TC TT TC TC TC CC CC CC TT TC TT TT 0 100.0%BB24662 TC TT TC TC TC CC CC CC TT TC TC TT 0 100.0% BB24663 TC TT TC TCTC CC CC CC TT TC TT TT 0 100.0% BB24664 TC TT TC TC TC CC CC CC TT TCTT TT 0 100.0% BB24665 TT FL FL CC TC FL CC FL TT TC CC TC 4 66.7%BB24666 TC TT TC FL TC CC CC CC TT TT TT TC 1 91.7% BB24667 TC TT TC TCCC CC CC CC TT TC TC TC 0 100.0% BB24668 TC TT TC TC CC CC CC CC TT TTTT TC 0 100.0% BB24669 TC TT TC TC CC TC CC CC TT CC TC CC 0 100.0%BB24670 TC TT TC TC CC CC CC CC TT TC TT TC 0 100.0% BB24672 TC TT TC TCTC CC CC CC TT TC TC CC 0 100.0% BB24673 TC TT TC TC TC CC CC CC TT TCTC TC 0 100.0% BB24674 TC TT TC TC CC CC CC CC TT TT TC CC 0 100.0%BB24675 TC TT TC TC TC CC CC CC TT TC TT TT 0 100.0% BB24676 TC TT TC FLTC CC CC CC TT TC TC TC 1 91.7% BB24678 TT FL CC CC TC CC CC CC TT TC TTTC 1 91.7% BB24679 TC FL TC TC TC CC CC CC TT CC TT TT 1 91.7% BB24680TC TT TC TC CC CC CC CC TT TT TT TT 0 100.0% BB24681 TC TT TC TC TC CCCC CC TT TC TT TT 0 100.0% BB24682 TC TT TC TC TC CC CC CC TT CC TT TT 0100.0% BB24683 TT FL CC CC TC CC CC CC TT TC TC TT 1 91.7% BB24684 TC TTTC TC CC CC CC CC TT TC TC TC 0 100.0% BB24685 TC TT TC TC CC CC CC CCCC TT TT CC 0 100.0% BB24686 TC TT TC TC CC CC CC CC TT CC TC TC 0100.0% BB24687 TC TT TC TC TC CC CC CC TT CC TC TT 0 100.0% BB24688 TTFL CC CC TC CC CC CC TT TC TT TT 1 91.7% BB24689 TC TT TC TC CC CC CC CCTT TC TT TC 0 100.0% BB24690 TC TT TC TC CC CC CC CC TT CC TC CC 0100.0% BB24691 TC TT TC TC CC CC CC CC TT TT CC CC 0 100.0% BB24692 TCTT TC TC CC CC CC CC TT TC TT TC 0 100.0% BB24693 TC TT TC TC TC CC CCCC TT CC TT TC 0 100.0% BB24694 TT FL CC CC TC CC CC CC TT TC TT TC 191.7% BB24695 TT FL CC CC TC CC CC CC TT TT TT CC 1 91.7% BB24696 TC TTTC TC CC CC CC CC TT TT TT TC 0 100.0% BB24697 TC TT TC TC TC TC CC CCTT TT TT TT 0 100.0% BB24698 TC TT TC TC CC CC CC CC TT TT TT TC 0100.0% BB24699 TC TT TC TC TC CC CC CC TT TC TT TC 0 100.0% BB24700 FLFL FL FL FL FL FL FL FL FL FL FL 12 0.0% BB24701 TT FL CC CC TC CC CC CCTT TT TT CC 1 91.7% BB24702 TT FL CC CC TC CC CC CC TT TC TC TC 1 91.7%BB24703 TT FL CC CC TC CC CC CC TT TC TT CC 1 91.7% BB24704 TC TT TC FLTC CC CC CC TT CC TT TC 1 91.7% BB24705 TC TT TC TC TC CC CC CC TT TC TTTT 0 100.0% BB24706 TT FL FL CC TC CC CC CC TT CC TT TC 2 83.3% BB24707TC TT TC TC CC CC CC CC TT TC TT TT 0 100.0% BB24708 TC TT TC FL TC CCCC CC TT TC TC TC 1 91.7% BB24709 TC TT TC TC TC CC CC CC TT TT TT TT 0100.0% BB24710 TC TT TC TC CC CC CC CC TT TC TT TC 0 100.0% BB24711 TCFL TC TC TC TC CC CC TT TC TT CC 1 91.7% BB24712 TC TT TC TC TC CC CC CCTT TC TT TC 0 100.0% BB24713 TC FL TC FL TC CC CC CC TT CC TT TT 2 83.3%BB24714 TT FL CC CC TC CC CC CC TT TC TT TT 1 91.7% BB24715 TT FL CC CCTC CC CC CC TT TC TT TC 1 91.7% BB24716 TC TT TC TC CC CC CC CC TT TC TCCC 0 100.0% BB24717 TC TT TC TC TC CC CC CC TT TC TT TC 0 100.0% BB24718TC TT TC TC TC CC CC CC TT TC TC TT 0 100.0% BB24719 TC TT TC TC TC CCCC CC TT CC TT CC 0 100.0% BB24720 TC TT TC TC CC CC CC CC TT TC TT TC 0100.0% BB24721 TC TT TC TC TC CC CC CC CC TT TT TC 0 100.0% BB24722 TCTT TC TC TC CC CC CC TT CC TT CC 0 100.0% BB24723 TC TT TC TC CC CC CCCC TT TT TC CC 0 100.0% BB24724 TC TT TC TC TC CC CC CC TT TC TT TT 0100.0% BB24725 TT FL CC CC TC CC CC CC TT CC TT CC 1 91.7% BB24726 TC TTTC TC TC CC CC CC TT TT TC TT 0 100.0% BB24727 TC TT TC TC CC CC CC CCTT TT TC TC 0 100.0% BB24728 TC TT TC TC TC CC CC CC TT TC TT TC 0100.0% BB24729 TC TT TC FL TC CC CC CC TT CC TT CC 1 91.7% BB24730 TC FLTC TC CC CC CC CC TT TT TC TT 1 91.7% BB24731 TC TT TC TC CC CC CC CC TTTT TC TC 0 100.0% BB24732 TT FL CC CC TC CC CC CC TT TT TT CC 1 91.7%BB24733 TC TT TC TC TC CC CC CC TT TC TT TC 0 100.0% BB24734 TC TT TC TCCC TC CC CC TT TC TT TC 0 100.0% BB24735 TT FL CC CC TC CC CC CC TT TTTT TT 1 91.7% BB24736 TC TT TC TC TC TC CC CC TT TC TT TT 0 100.0%BB24737 TC TT TC FL TC CC CC CC TT TT TT TT 1 91.7% BB24738 TT FL CC CCTC CC CC CC TT TC TC CC 1 91.7% BB24739 TT FL CC CC TC CC CC CC TT TC TCTC 1 91.7% BB24740 FL FL FL FL FL FL FL FL FL FL FL FL 12 0.0% BB24741TC TT TC TC TC CC CC CC TT TC TT TC 0 100.0% BB24742 TC TT TC TC TC CCCC CC TC TT TT TT 0 100.0% BB24743 TC TT TC TC TC CC CC CC TT TT TT TT 0100.0% BB24744 TC TT TC TC TC CC CC CC TT TT TC TC 0 100.0% BB24745 TCTT TC TC TC CC CC CC TT CC TT TT 0 100.0% BB24746 TC TT TC TC TC CC CCCC TT TC TT TC 0 100.0% BB24747 TC TT TC TC CC CC CC CC TT TC TT TT 0100.0% BB24748 TC TT TC TC TC CC CC CC TT TC TT TC 0 100.0% BB24749 TTFL CC CC CC CC CC CC TT TC TT TT 1 91.7% BB24750 TC TT TC TC TC CC CC CCTT TC TT TC 0 100.0% BB24751 TC TT TC TC TC CC CC CC TC TC TT TC 0100.0% BB24752 TC TT TC TC TC CC CC CC TT TT TC TC 0 100.0% BB24753 FLFL FL FL FL FL FL FL FL FL FL FL 12 0.0% BB24754 TC TT TC TC CC CC CC CCTT TC TC FL 1 91.7% BB24755 TT FL CC CC TC CC CC CC TT TC TT TT 1 91.7%BB24756 TC FL TC TC CC CC CC CC TT TT TC TT 1 91.7% BB24757 TC TT TC FLTC CC CC CC TT TT TT TC 1 91.7% BB24758 TC TT TC TC TC CC CC CC TT CC TCTC 0 100.0% BB24759 TC TT TC TC TC CC CC CC TT CC TT TC 0 100.0% BB24760TC TT TC TC TC CC CC CC TT TC TC TC 0 100.0% BB24761 TC TT TC TC CC CCCC CC TT TC TT CC 0 100.0% BB24762 TC TT TC TC TC CC CC CC TT TC TT TC 0100.0% BB24763 TC TT TC TC FL TC CC CC TT CC FL TC 2 83.3% BB24764 TT FLCC TC TC CC TC CC TT TT TT CC 1 91.7% BB24765 TC FL TC TC CC CC CC CC TTTT TT CC 1 91.7% BB24766 TC TT TC FL TC CC CC CC TT TC TT TC 1 91.7%BB24767 TC TT TC TC TC CC CC CC TT CC TT TC 0 100.0% BB24768 TC TT TC FLTC CC CC CC TT TT TT TC 1 91.7% BB24769 TC TT TC TC CC CC CC CC TT CC TTCC 0 100.0% BB24770 TT FL CC CC CC CC CC CC TT CC TT CC 1 91.7% BB24771TC TT TC TC TC CC CC CC TT TC TT TC 0 100.0% BB24772 TC TT TC TC TC CCCC CC TT TC TT TT 0 100.0% BB24773 TC TT TC TC TC CC CC CC TT TT TC TC 0100.0% BB24774 TC TT TC TC CC CC CC CC TT CC TT TC 0 100.0% BB24775 TCTT TC TC TC CC CC CC TT CC TT CC 0 100.0% BB24776 TC TT TC TC TC CC CCCC TT TT TT TC 0 100.0% BB24777 TC TT TC TC TC CC CC CC TT TC TT TT 0100.0% BB24778 TC TT TC TC CC CC CC CC TT CC TT TC 0 100.0% BB24779 TCFL TC TC CC CC FL CC TT TT TT TT 2 83.3% BB24780 TC TT TC TC TC CC CC CCTT TC TC CC 0 100.0% BB24781 TC TT TC TC TC CC CC CC TT TC TC CC 0100.0% BB24782 TC TT TC TC TC CC CC CC TT CC TT CC 0 100.0% BB24783 TTFL CC CC TC CC CC CC TT TC TC TC 1 91.7% BB24784 TC TT TC TC TC CC CC CCTT CC TT TC 0 100.0% BB24785 TC TT TC TC TC CC CC CC TT TC TT TC 0100.0% BB24786 TC TT TC TC TC CC CC CC TT TT TT TC 0 100.0% BB24787 TCTT TC TC TC CC CC CC TT TT TC TC 0 100.0% BB24788 TC TT TC TC CC CC CCCC TT TT TC TC 0 100.0% BB24789 TC TT TC TC TC TC CC CC TT TT TT TT 0100.0% BB24790 TT FL CC CC TC CC CC CC TC TT TT TC 1 91.7% BB24791 TT FLCC FL TC CC FL CC TT TC TT CC 3 75.0% BB24792 TC TT TC TC CC TC CC CC TTTT TT TC 0 100.0% BB24793 TT FL CC CC FL CC CC CC TT TT TT TC 2 83.3%BB24794 TC TT TC TC CC CC CC CC TT CC TC CC 0 100.0% BB24795 TC TT TC TCTC CC CC CC TT CC TT TC 0 100.0% BB24796 TC TT TC TC TC CC CC CC TT TCTT TC 0 100.0% BB24797 TC TT TC TC TC CC CC TC TT TT TT TT 0 100.0%BB24798 TC TT TC TC TC CC CC CC TT CC TT TC 0 100.0% BB24799 TC TT TC TCTC CC CC CC TT TC TT CC 0 100.0% BB24800 TC TT TC TC TC CC CC CC TT TCTT TC 0 100.0% BB24801 FL FL FL FL FL FL FL FL FL FL FL FL 12 0.0%BB24803 TC TT TC TC CC CC CC CC TT CC TT FL 1 91.7% BB24804 TT FL CC CCTC CC CC CC TT TC TT CC 1 91.7% BB24805 TC TT TC TC CC CC CC CC TT TT TTTT 0 100.0% BB24806 TC TT TC TC CC CC CC CC TT TC TT CC 0 100.0% BB24807TC TT TC FL TC CC CC CC TC TT TT CC 1 91.7% BB24808 TC TT TC TC TC CC CCCC TT TC TT TC 0 100.0% BB24809 TC TT TC TC TC CC CC CC TT TC TC CC 0100.0% BB24810 TC TT TC TC CC CC CC CC TT TC TT TT 0 100.0% BB24811 FLFL FL FL FL FL FL FL FL FL FL FL 12 0.0% BB24812 TC TT TC TC TC CC CC CCTT TC TT CC 0 100.0% BB24815 TC TT TC TC TC CC CC CC TT TT TC TC 0100.0% BB24817 TC TT TC TC TC CC CC CC TT CC TT TT 0 100.0% BB24818 TCTT TC TC TC CC CC CC TT TC TT CC 0 100.0% BB24819 TC TT TC TC CC CC CCCC TT CC TT TC 0 100.0% BB24820 TC TT TC TC TC CC CC CC TT CC TC CC 0100.0% BB24821 TT FL CC CC TC CC CC CC TT CC TT TT 1 91.7% BB24823 TC TTTC TC TC TC CC CC TT TC TC TT 0 100.0% BB24824 TC TT TC TC TC CC CC CCTT TT TC TT 0 100.0% BB24826 TC TT TC TC TC CC CC CC TT TC TT TC 0100.0% BB24827 TT FL CC TC TC CC CC CC TT CC TT TT 1 91.7% BB24830 TC TTTC TC TC CC CC CC TT CC TT TC 0 100.0% BB24831 TC TT TC TC CC CC CC CCTT CC TT TC 0 100.0% BB24832 TT FL CC CC TC CC CC CC TT TT TT TC 1 91.7%BB24833 TC TT TC TC TC CC CC CC TT TC TC TC 0 100.0% BB24834 TC TT TC TCCC CC CC CC TT TC TT CC 0 100.0% BB24836 TC TT TC TC TC CC CC CC TT TTTT CC 0 100.0% BB24837 TC TT TC TC TC CC CC CC TT TC TT TT 0 100.0%BB24838 TC TT TC TC TC CC CC CC TT CC TT TC 0 100.0% BB24839 TC TT TC TCCC TC CC CC TT TC TT CC 0 100.0% BB24841 TC TT TC TC TC CC CC CC TT TTTT TC 0 100.0% BB24842 TC TT TC TC TC CC CC CC TT TC TC TT 0 100.0%BB24843 TT FL FL TC FL FL CC FL TT FL TC TC 6 50.0% BB24844 FL FL FL FLFL FL FL FL FL FL FL FL 12 0.0% BB24847 TC TT TC TC CC TC CC CC TT TT TTTT 0 100.0% Q1H2O FL FL FL FL FL FL FL FL FL FL FL FL 12 0.0% Q2H2O FLFL FL FL FL FL FL FL FL FL FL FL 12 0.0% Q3H2O FL FL FL FL FL FL FL FLFL FL FL FL 12 0.0% Q4H2O FL FL FL FL FL FL FL FL FL FL FL FL 12 0.0%RHD4 RHD7 RHD9 RHCE2 RHCE5 KEL6 KEL8 Sample FL 15 86 20 54 23 18 17Sample Pass 357 286 352 318 349 354 355 Call Rate 95.97% 76.88% 94.62%85.48% 93.82% 95.16% 95.43% Genotypes (N) XX (TT) 64 286 0 0 0 0 0 XY(TC) 293 0 293 260 246 28 1 YY (CC) 0 0 59 58 103 326 354 Allele Freq X(p) 58.96% 100.00% 41.62% 40.88% 35.24% 3.95% 0.14% Y (q) 41.04% 0.00%58.38% 59.12% 64.76% 96.05% 99.86% DI18 FYP FY2 GP3A JK9 Sample FL 17 1516 16 17 Sample Pass 355 357 356 356 355 Call Rate 95.43% 95.97% 95.70%95.70% 95.43% Genotypes (N) XX (TT) 0 348 112 263 87 XY (TC) 2 7 155 89178 YY (CC) 353 2 89 4 90 Allele Freq X (p) 0.28% 98.46% 53.23% 86.38%49.58% Y (q) 99.72% 1.54% 46.77% 13.62% 50.42%

1-19. (canceled)
 20. A probe comprising (i) at least one of thenucleotide sequence of nucleotide positions 1 to 20 of any one of SEQ IDNO: 25 to 35 and nucleotide position 1 to 18 of SEQ ID NO: 6 and (ii)the nucleotide sequence of nucleotide positions 21 to 45 of as set forthin SEQ ID NO:
 31. 21. The probe of claim 20 for use as an extensionprobe for the detection of a T/C SNP in an exon 8 of a KEL gene.
 22. Ablood group/platelet antigen typing kit comprising a firstoligonucleotide having a nucleotide sequence as set forth in SEQ ID NO:13, a second oligonucleotide having a nucleotide sequence as set forthin SEQ ID NO: 14 and a probe having the nucleotide sequence as definedin claim
 1. 23. A method of detecting a Kp^(a/b) blood group antigen ina sample, said method comprising: (a) providing genomic DNA from saidsample; (b) submitting the genomic DNA of step (a) to a PCRamplification with at the first oligonucleotide as defined in claim 3and the second oligonucleotide as defined in claim 22 to obtain at leastone amplification product; and (c) analyzing the at least oneamplification product of step (b) to detect the blood group antigen inthe sample.
 24. The method of claim 23, wherein the at least oneamplification product is digested with a restriction enzyme.
 25. Themethod of claim 24, wherein said restriction enzyme is Exonuclease I orshrimp alkaline phosphatase.
 26. The method of claim 23, furthercomprising, in step (c), generating at least one extension product fromthe at least one amplification product.
 27. The method of claim 26,further comprising hybridizing the at least one extension productionwith a probe having the sequence of nucleotide positions 21 to 45 of asset forth in SEQ ID NO:
 31. 28. The method of claim 27, wherein theprobe has the sequence as defined in claim
 1. 29. The method of claim27, wherein said extension product is hybridized to a tag-arrayedmicroplate.